|dc.description.abstract||The fluorescent protein phosphatase inhibition assay (PPINA), modified into a prebiosensor design that utilises a micro-filter plate, was assessed on its performance to detect okadaic acid (OA), the major diarrhetic shellfish poisoning (DSP) toxin, and microcystins (MC), using two enzymes, protein phosphatase type 2A (PP-2A) and a recombinant protein phosphatase type 1 (R.PP-1). The pre-biosensor design consisted of immobilising the enzyme prior to conducting the assay, so that the enzyme could be retained for re-use. Modifications were made to the immobilised PP-2A and R.PP-1 method to maximise the activity and stability of the enzyme. These modifications involved adjusting cofactors in the assay, altering pH and adjusting components in the buffers during the immobilisation procedure. Both of these enzymes showed high activity in the optimised, immobilised system and were stable over a long period of time (600 hours).
The feasibility of the immobilised PPINA for use as a biosensor, for MC and OA detection, was determined by constructing dose-response curves for each toxin with PP-2A and R.PP-1. Under the optimised, immobilised assay conditions, OA and MC-LR inhibited PP-2A dose-dependently, with IC₅₀ values of 55 and 80 nM, respectively. Similarly, immobilised R.PP-1 was inhibited by MC dose-dependently, with an IC₅₀ value of 150 nM, under optimised assay conditions. This compares with IC₅₀ values of 5.5 and 3.2 nM for PP- 2A against OA and MC-LR respectively, and 0.9 nM for R.PP1 against MC-LR, in the standard, unimmobilised PPINA format. Thus, the sensitivities of the immobilised enzymes were at least 10-fold less for MC-LR and OA detection, compared to the unimmobilised PPINA format. These detection limits for the immobilised PP-2A and R.PP-1 enzymes were outside the proposed guidelines of 1 µg of MC-LR/L for drinking water set by the WHO, and 160 µg of OA equivalents/kg in mussel meat set by the European Commission on Standards. Furthermore, R.PP-l was not suitable for detecting OA, as immobilised and unimmobilised enzyme activity was induced (rather than inhibited) between 10-200 nM and 1-100 nM of OA, respectively. PP-2A is the preferred enzyme over R.PP-1 for OA and MC detection, because of the greater sensitivity of this enzyme to both toxins.
The immobilised enzyme assay system (using PP-2A and R.PP-1) was tested for re-usability, by determining if the toxin inhibitors (i.e. OA and MC-LR) can be removed from the system by washing with Tris buffer (PH 7.0). Removal of the toxins should result in enzyme activity being restored in the assay to pre-toxin levels, which would render the assay suitable for repeated use. The immobilised PP-2A enzyme was not suitable for re-use, as permanent binding occurred between the enzyme and both toxins. Consequently, this prevents the use of non-replaceable PP-2A enzyme in a biosensor format. In contrast, R.PP-1 showed some potential suitability in this format for MC detection, as enzyme activity was restored to pre-toxin levels after 20 washes.
The immobilised R.PP-1 and PP-2A enzyme assays were applied to the analysis of algal and mussel samples naturally contaminated with MCs and DSP toxins, respectively. This was carried out to compare the sensitivity of the immobilised PPINA with LC-MS and other PPINA methods. The mussel and algal tests gave an insight into the applicability of the pre-biosensor method. Firstly, matrix effects, caused by confounding substances in sample extracts, were shown to interfere with the enzyme-toxin binding. Secondly, sample dilution was found to be important to maximise toxin detection. Further studies to address these issues are discussed.
This study is the first to assess the suitability of the immobilised PPINA method for translation into a prototype biosensor format. The results indicate that while PP-2A is the preferred enzyme because of its enhanced sensitivity over RPP-1, it is not amenable for reuse in a biosensor format. Nevertheless, the enzyme could be used in a biosensor as a disposable component. However, further improvements to enhance sensitivity to the toxins are necessary before translation to a biosensor format is considered. This study is also the first to demonstrate the use of the fluorimetric substrate, MUMP for the R.PP-1 enzyme assay.||en