714 K. Campbell et al. Figure 2. SPR optical biosensor sensorgram of the molecular interaction between the binder and immobilised surface. Source: Abery (2001). Courtesy: Biacore International AB, GE Healthcare. surface established the baseline value and as the sample solution passed over the sensor surface, the profile of the molecular interaction appeared. During injection the observed binding rate was the difference between the molecules binding to the surface and molecules leaving the surface. Consequently, binding appears fastest at the begin- ning of the injection as the binder in the sample readily interacts with the high concentration of available toxin-binding partners on the sensor sur- face. As the injection continues, the observed rate of binding decreased as fewer toxin molecules are available on the surface. After the injection was complete, the binder dissociated and was washed away by the continuous flow of buffer. The sensor surface was regenerated back to the baseline value for the analysis of the next sample (Campbell et al. 2007). The SPR optical biosensor method for PSP toxins was designed as an inhibition assay. A set amount of PSP binder was mixed with the sample and injected over the surface of a sensor chip to which toxin was immobilised. When no PSP toxins were present in the sample, the binder was then free to bind to the immobilised toxin producing a response (Figure 3a) (Campbell et al. 2007). When PSP toxins were present in the sample, these then bound to the binder, which inhibited the binder from binding to the immobilised toxin on the chip surface and a lower response was produced (Figure 3b) (Campbell et al. 2007). For most biomolecules the change in response is proportional to the mass of the material that has bound to the surface. Therefore, for binders to elicit a change in response from the baseline, they must be greater than 1000 daltons and hence this is why the low molecular weight toxins are immobilised to the surface. Using this assay format, the level of binding to the surface was inversely proportional to the concentration of PSP toxins present in the sample. Figure 4 shows a typical response against a concentration calibration curve. Further developments in optical SPR biosensor technology, particularly the progression of multichan- nel and multiplex instruments such as the Biorad Proteon XPR36 (http://www.bio-rad.com) or Biacore prototype developed as part of the European Union 6th Framework Project BioCop (http://www.biocop. org), provide additional options for dealing with toxicity factors of toxins and cross-reactivity issues of binders. Within the European Union 7th Framework programme the project CONffIDENCE is evaluating the BioCop multiplex optical SPR for developing a single assay for multiple toxin groups legislated by the European Union and emerging toxins to European Union waters (http://www.conffidence.eu). This mul- tiplex prototype device is a four-channel instrument with four detection spots within each channel allow- ing for the simultaneous analysis of up to 16 differ- ent analytes in a sample. This increases the scope of binder interactions that could be employed to detect the different PSP analogues. An ideal scenario would be to have a highly specific binder for each of the main PSP toxin groups. Each binder assay for each group could be isolated in separate channels for simultaneous detection in a sample, and then not only would the analyst be able to determine if PSP toxin was present, but also to which group it belonged and a correlation made of the toxic potency level for that sample. Binders for SPR analysis of PSP toxins A number of different types of binder have been exploited in the development of various methodologies for the detection of PSP toxins. These include receptors such as the sodium channel receptor (SCR) and saxiphilin receptor, polyclonal and monoclonal anti- bodies raised against various toxin analogues and chemical binders (Llewellyn and Doyle 2001; Usleber et al. 2001; Gawley et al. 2007; van Dolah et al. 2009). The general advantages and disadvantages of binders that have been previously investigated in the develop- ment of assays for SPR analysis for PSP toxins are highlighted in Table 1. Receptors Receptors can be either cell membrane bound or cytoplasmic proteins that may be exploited for their ligand-binding capabilities for screening methodologies based on their functional response. SCRs from crude rat brain membrane preparations have been used in radioligand binding assays (Wiegele and Barchi 1978) and in recent years they have been configured into a microtitre plate format (Powell and Doucette 1999;