Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor
Introduction
The safety and quality of the world’s coastal areas is an important issue because they harbour complex ecosystems and represent an important economic source with regards to tourism, fishery and aquaculture. However, blooms of microalgae that are potentially harmful with respect to the ecosystem, public health or economic aspects frequently affect coastal areas. Harmful algae can be classified into three groups. The first group is not toxic, nevertheless, it has the potential to harm the environment by forming dense blooms that can cause an oxygen-depletion that results in indiscriminate kills of fish and other organisms. Toxic algae are assigned to a second group of harmful algae that threaten the environment by the production of very potent toxins, which can cause both animal and human poisoning. The third group of harmful algae comprises those microalgae that either harm fish mechanically or by the production of haemolytic substances (Hallegraeff, 1993). There are about 300 species known to have the potential to form algal blooms and approximately 85 of theses species are potential toxin producers (Sournia et al., 1991, IOC, 2002). The majority of the known toxic algae species belongs to three divisions: Cyanophyta, Dinophyta and Haptophyta (Fogg, 2002). However, the division Dinophyta contains the largest number of harmful algal species (Taylor, 1985). Among the Dinoflagellates there are about 20 species known to produce potent toxins, for example, saxitoxins that cause the life threatening syndrome paralytic shellfish poisoning (PSP). PSP is caused by the consumption of contaminated shellfish (Hallegraeff, 1993). The dinoflagellate Alexandrium ostenfeldii produces PSP-toxins (Hansen et al., 1992) and other toxins. Recently, new toxins, including the spirolides, produced by the marine dinoflagellate A. ostenfeldii have been isolated. Mammalian toxicity of spirolides was confirmed by mouse bioassays. Spirolides proved to be highly toxic in intraperitoneal injections of lipophilic, contaminated shellfish extracts in mice, including neurological symptoms, followed by rapid death (Cembella, 1998, Cembella et al., 2000, Hu et al., 2001). The dinoflagellate A. ostenfeldii is cosmopolitan, it has been observed in the waters of Greenland (Hansen-Ostenfeld, 1913), Norway (Balech and Tangen, 1985), Spain (Fraga and Sanchez, 1985), Denmark (Moestrup and Hansen, 1988), Russia (Konovalova, 1991), Egypt (Balech, 1995), Canada (Cembella et al., 1999) and New Zealand (McKenzie et al., 1996).
In the light of the threats by the toxic algae that occur at the coastlines all over the world (Hallegraeff, 1995) numerous monitoring programs have been launched that observe the phytoplankton composition on a regular basis. This is done not only to initiate testing of toxin in shellfish, but also to serve as an early warning system for fish-farms to avoid economic damage caused by the bloom of a toxic algae that has the potential to kill the caged fish. With regard to this the European Union requires that the member states monitor the water at their coastlines for toxin-producing plankton and toxins in mussels (Directive 91/492d/EC and Commision Decision 2002/225/EC). Monitoring is very labour intensive and costly. It requires the analysis of large numbers of samples. Currently, the identification of phytoplankton cells is done by taxonomy, which is based on a broad expertise of specially trained staff, expensive equipment like electron microscopes and is very time consuming. Therefore, a more rapid, secure and inexpensive method would be welcome by all monitoring programs. In this respect the application of DNA-biosensors could serve the needs of monitoring programs. DNA-biosensors are known from various areas of interest that take advantage of the hybridisation principle e.g., in the face of the mailings of letters containing Bacillus anthracis in fall of 2001 a biosensor for the specific identification of the bacterium was developed (Hartley and Baeumner, 2003). The identification of organisms with a DNA-biosensor is based on specific probes that target DNA-sequences that are only present in the organism of interest. In the past decade, numerous probes have been developed for the identification of phytoplankton and toxic algae, respectively (Scholin and Anderson, 1998, Simon et al., 2000). Here, we present the adaptation of a DNA-biosensor for the electrochemical detection of the toxic dinoflagellate A. ostenfeldii with molecular probes. The technical background of the device was presented in detail previously to the public in the German patent application DE 10032 042 A1 (Elektrochemischer Einwegbiosensor für die quantitative Bestimmung von Analytkonzentrationen in Flüssigkeiten). The system is based on two major parts. The first part is a disposable sensor chip and a handheld device for the measurement of the electrochemical signal. The disposable sensor chip consisting of a carrier material harbours a working electrode, on which the detection reaction takes place, a reference electrode and an auxiliary electrode. The working electrode has a diameter of 1 mm and consists of a carbon paste. A biotinylated probe is immobilised in the reaction layer of the working electrode via avidin. The nucleic acids are detected on the sensor chip via a sandwich-hybridisation (Zammatteo et al., 1995, Rautio et al., 2003). The underlying principle of this method is that one target specific probe, here called capture probe, is immobilised on the surface of the working electrode. If a target nucleic acid is bound to the immobilised probe on the working electrode, the detection of the nucleic acid takes place via a hybridisation to a second target specific probe, which is coupled to digoxygenin that is recognized by a digoxigenin specific antibody. The antibody in turn is coupled to horseradish-peroxidase that catalyses the reduction of hydrogen peroxide (substrate of the horseradish-peroxidase) to water. The reduced peroxidase is regenerated by p-aminodiphenylamin (ADPA), that functions as a mediator. The oxidised mediator gets reduced at the working electrode with a potential of −150 mV (versus Ag/AgCl) (Fig. 1). In this set up it is only possible to measure an electrochemical signal, if the target nucleic acid as the link between the two probes is present in the system.
Section snippets
Algal strains
The specificity of the probes targeting the 18S rRNA of A. ostenfeldii was tested with the following strains: A. ostenfeldii K0287 and K0324 (Scandinavian Culture Centre for Algae and Protozoa, University of Copenhagen), A. ostenfeldii BAH ME 136 (Biologische Anstalt Helgoland, Germany), Alexandrium minutum AL3T (Gulf of Trieste, Italy, A. Beran), Alexandrium lusitanicum BAH ME91 (Biologische Anstalt Helgoland, Germany), Alexandrium pseudogonyaulax AP2T (Gulf of Trieste, Italy, A. Beran),
Probe design
For the sandwich-hybridisation two probes that specifically bind to the 18S-rRNA of A. ostenfeldii (Table 1) have been designed using the ARB software package (Ludwig et al., 2004). The probes have at least two mismatches against all non-target organisms listed in the ARB database. The overall specificity of the probes was tested by doing a BLAST search (Altschul et al., 1990) against all public available sequences. To avoid possible effects of degradation of the target nucleic acid the chosen
Discussion
Here, we present a chip-based electrochemical biosensor for the identification of the toxic dinoflagellate A. ostenfeldii via a sandwich hybridisation on molecular level. The biosensor is an inexpensive, easy and rapid technology for the identification of phytoplankton. Electrochemical readings of the handheld device and the protocols are unambiguous and even for a scientific layperson easy to use and interpret. Therefore, this system has great potential to serve as an alternative to
Conclusion
A DNA-biosensor was adapted to the electrochemical detection of the toxic dinoflagellate A. ostenfeldii and the sensitivity of the DNA-biosensor was increased by an optimisation of the hybridisation conditions. The electrochemical DNA-biosensor presented in this publication is a proof of principle for a DNA-handheld device that possesses the potential to serve as a quick and easy method for the identification of toxic algae. The device could facilitate the work that must be done in the course
Acknowledgements
We would like to thank Inventus Biotec, Germany, for excellent cooperation with regard to the allocation of the handheld device, the disposable sensor chips and the adaptation of the glucose sensor chips to our needs, as well as for valuable discussion.
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