Sonja Diercks

Development and adaptation of a multiprobe biosensor for the use in a semi-automated device for the detection of toxic algae

Worldwide monitoring programs have been launched for the observation of phytoplankton composition and especially for harmful and toxic microalgae. Several molecular methods are currently used for the identification of phytoplankton but usually require transportation of samples to specialised laboratories. For the purpose of the monitoring of toxic algae, a multiprobe chip and a semi-automated rRNA biosensor for the in-situ detection of toxic algae were developed. Different materials for the electrodes and the carrier material were tested using single-electrode sensors and sandwich hybridisation that is based on species-specific rRNA probes. Phytoplankton communities consist of different species and therefore a biosensor consisting of a multiprobe chip with an array of 16 gold electrodes for the simultaneous detection of up to 14 target species was developed. The detection of the toxic algae is based on a sandwich hybridisation and an electrochemical detection method.

Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces

Biosensors and microarrays are powerful tools for species detection and monitoring of microorganisms. A reliable identification of microorganisms with probe-based methods requires highly specific and sensitive probes. The introduction of locked nucleic acid (LNA) promises an enhancement of specificity and sensitivity of molecular probes. In this study, we compared specificity and sensitivity of conventional probes and LNA modified probes in two different solid phase hybridisation methods: sandwich hybridisation on biosensors and on DNA microarrays. In combination with DNA-microarrays, the LNA probes displayed an enhancement of sensitivity, but also gave more false-positive signals. With the biosensor, the LNA probes showed neither signal enhancement nor discrimination of a single mismatch. In all cases, conventional DNA probes showed equal or better results than LNA probes. In conclusion, LNA technology may have great potential in methods that use probes in suspension and in gene expressions studies, but under certain solid surface-hybridisation applications, they do not improve signal intensity.

Detection of Phytoplankton with Nucleic Acid Sensors

A potential hand-held biosensor system for the in-situ analysis of toxic algae was developed during the EU-project ALGADEC. Identification of toxic algae is based on molecular probes that specifically target its rRNA. 17 taxon specific probe sets were developed for harmful algae that occur in three different coastal areas in Europe. A sandwich-hybridization and two labelled probes are used to detect the rRNA. A capture probe, immobilised on the biosensor, binds to RNA-strands isolated from the target organism. A second digoxigen-labelled probe binds also to the RNA-strands. After incubation with an antibody-enzyme complex directed against digoxigenin, a substrate is added and a redox-reaction takes place. The resulting electrical current is measured and the amount of bound rRNA is proportional to the electrical current. The adaptation to the sensor and the probe specificity tests were done using laboratory strains with closely related species to avoid false positives and to guarantee that only desired strains are detected. The signals from the different probes are recorded by a microcontroller unit. If a PC is connected to the system, an easy to operate software visualizes process data, graphic results, and the measured values will be stored on the hard disc. The main steps of the analysis process are executed automatically in the measurement device. Only a manual filtering, including a lysis procedure has to be done before the automatic measurement. The portable ALGADEC device is also capable to operate as a stand-alone system with

Automated nucleic biosensors - a key to high resolution monitoring of marine phytoplankton

Changes in plankton community structures in response to climate change and the climatic sensitivity of species are currently driving topics in marine research. In the marine environment phytoplankton consists of major primary producers, but also harmful algae that can negatively influence marine ecosystems. It is expected that climate related environmental change could result in changes in the abundance, spatial distribution, biogeography or dominance of phytoplankton species. In order to evaluate consequences of climate change for marine ecosystems it is necessary to possess high resolution information in time and space on current abundances and patterns within the phytoplankton. However, the generation of these data is constrained by a variety of reasons like the size or insufficient morphological markers of the taxa and the costs to provide samples with high spatiotemporal resolution. In the past decade the application of biosensor technology has gained significant impact in respect to microbial analysis. More and more publications describe the development of molecular sensors dedicated to the detection of microbial organisms. In the EU FP6- project ALGADEC a portable semi-automated biosensor-system has been developed in order to facilitate the detection of toxic algae in the field. This device enables the electrochemical detection of microalgae from watersamples in less than two hours, without the need of expensive equipment. This device is a prototype device that serves as a cornerstone of a new molecular based strategy for the monitoring of phytoplankton. Currently we are working on the adaptation of the biosensor to the surveillance of key species in the North Sea. And, we aim at a full automation of the system in order to provide an autonomous monitoring tool for phytoplankton. In the Future an autonomous biosensor can be combined with present in situ measurement systems for the marine environment like the FerryBox-system of the GKSS. This would serve the need for technologies that allow high resolution monitoring of marine phytoplankton in order to evaluate consequences of environmental change in the oceans.