Okadaic acid (OA), a lipophilic toxin, is produced by Dinophysis and Prorocentrum, and causes diarrheic shellfish poisoning to humans. The mechanism of OA action is based on the reversible inhibition of protein phosphatase type 2A (PP2A) by the toxin. Therefore, this inhibition could be used to develop assay for OA detection. In this work, a colorimetric test based on the PP2A inhibition was developed for OA detection. PP2A from GTP and Millipore was immobilized on silica sol-gel, and the detection was performed. A limit of detection of 0.29 and 1.14 μg/L was respectively observed for enzyme from GTP and Millipore. The immobilization technique provided a tool to preserve the enzymatic activity, which is very unstable in solution. The PP2A immobilized sol-gel exhibited a storage stability of near 5 months, when microtiter plate with enzyme-immobilized polymer was kept at −18C°. The combination of the simplicity of the colorimetric method, along with long storage stability achieved by sol-gel immobilization, demonstrated the potentiality of this technique to be used for commercial purpose.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Steidinger, K. A. (1993). Algal toxins in seafood and drinking water. New York: Academic Press.
Yasumoto, T., & Murata, M. (1993). Marine toxins. Chemical Reviews, 93, 1897–1909.
Suganuma, M., Fujuki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., et al. (1988). Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proceedings of the National Academy of Sciences of the United States of America, 85(6), 1768–1773.
EFSA. (2008). Marine biotoxins in shellfish – okadaic acid and analogues Scientific Opinion of the Panel on Contaminants in the Food chain. The EFSA Journal, 589, 1–62.
Yasumoto, T., Oshima, Y., & Yamaguchi, M. (1978). Occurance of a new type of shellfish poisioning in the Tohuko district. Bulletin of the Japanese Society of the Science of Fish, 44, 1249–1255.
Kreuzer, M. P., O'Sullivan, C. K., & Guilbault, G. G. (1999). Alkaline phosphatase as a label for immunoassay using amperometric detection with a variety of substrates and an optimal buffer system. Analytica Chimica Acta, 393, 95–102.
Bialojan, C., & Takai, A. (1988). Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochemical Journal, 256, 283–290.
Takai, A., Ohno, Y., Yasumoto, T., & Mieskes, G. (1992). Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis. Biochemical Journal, 287, 101–106.
Campas, M., & Marty, J.-L. (2007). Enzyme sensors for the electrochemical detection of the marine toxin okadaic acid. Analytica Chimica Acta, 605, 87–93.
Canete, E., Campas, M., De Le Iglesia, P., & Diogene, J. (2010). NG108-15 cell-based and protein phosphatase inhibition assays as alternative semiquantitative tools for the screening of lipophilic toxins in mussels.okadaic acid detection. Toxicology in Vitro, 24, 611–619.
Ikehara, T., Imamura, S., Yoshino, A., & Yasumoto, T. (2010). PP2A inhibition assay using recombinant enzyme for rapid detection of okadaic acid and its analogs in shellfish. Toxins, 2, 195–204.
Simon, J. F., & Vernoux, J.-P. (1994). Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate. Natural Toxins, 2, 293–301.
Tubaro, A., Florio, C., Luxich, E., Sosa, S., Loggia, R. D., & Yasumoto, T. (1996). A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels. Toxicon, 34, 743–752.
Fágain, C. O. (2003). Enzyme stabilization—recent experimental progress. Enzyme and Microbial Technology, 33, 137–149.
Kim, J., Grate, J. W., & Wang, P. (2006). Nanostructures for enzyme stabilization. Chemical Engineering Science, 61, 1017–1026.
Roger, A. S. (2007). Enzyme immobilization: the quest for optimum performance. Advanced Synthesis and Catalysis, 349, 1289–1307.
Hart, J. P., & Wring, S. A. (1997). Recent developments in the design and application of screen-printed electrochemical sensors for biomedical, environmental and industrial analyses. Trends in Analytical Chemistry, 16, 89–103.
Li, Y.-G., Zhou, Y.-X., Jiang, J.-H., & Ma, L.-R. (1999). Immobilization of enzyme on screen-printed electrode by exposure to glutaraldehyde vapour for the construction of amperometric acetylcholinesterase electrodes. Analytica Chimica Acta, 382, 277–282.
Rouillon, R., Mionetto, N., & Marty, J.-L. (1992). Acetylcholine biosensor involving entrapment of two enzymes. Optimization of operational and storage conditions. Analytica Chimica Acta, 268, 347–350.
Noguer, T., Leca, B., Jeanty, G., & Marty, J.-L. (1999). Biosensors based on enzyme inhibition: Detection of organophosphorus and carbamate insecticides and dithiocarbamate fungicides. Field Analytical Chemistry & Technology, 3, 171–178.
Noguer, T., Balasoiu, A. M., Avramescu, A., & Marty, J.-L. (2001). Development of a disposable biosensor for the detection of metam-sodium and its metabolite mitc. Analytical Letters, 34, 513–528.
Brinker, C. J., & Sheerer, G. W. (1990). Sol-gel science. The physics and chemistry of sol-gel processing. San Diego: Academic Press.
Andreescu, S., Barthelmebs, L., & Marty, J.-L. (2002). Immobilization of acetylcholinesterase on screen-printed electrodes: comparative study between three immobilization methods and applications to the detection of organophosphorus insecticides. Analytica Chimica Acta, 464, 171–180.
Campas, M., Szydlowska, D., Trojanowicz, M., & Marty, J.-L. (2005). Towards the protein phosphatase–based biosensor for microcystin detection. Biosensors and Bioelectronics, 20, 1520–1530.
Li, F.-Y., Xing, Y.-J., & Ding, X. (2007). Immobilization of papain on cotton fabric by sol-gel method. Enzyme and Microbial Technology, 40, 1692–1697.
Desimone, M. F., De Marzi, M. C., Copello, G. J., Fernández, M. M., Pieckenstain, F. L., & Malchiodi, E. L. (2006). Production of recombinant proteins by sol-gel immobilized Escherichia coli. Enzyme and Microbial Technology, 40, 168–171.
Alvarez, G. S., Desimone, M. F., & Diaz, L. E. (2007). Immobilization of bacteria in silica matrices using citric acid in the sol-gel process. Applied Microbiolology and Biotechnology, 73, 1059–1064.
Bradbury, S.-L., & Jakoby, W.-B. (1972). Glycerol as an enzyme-stabilizing agent: Effects on aldehyde dehydrogenase. Proceedings of the National Academy of Science of the United States of America, 69, 2373–2376.
Nita, M., Raducan, A., Puiu, M., & Oancea, D. (2007). Stabilization of catalase in the presence of additives. Bucharest: ARS.
Eixarch, H., Garibo, D., Canete, E., De La Iglesia, P., Fernandez, M., Diogene, J., et al. (2010). Protein phosphatase and cell-based assays as toxicosurveillance tools: Matrix effects in the analysis of marine toxins present in shellfish. Toxicological Letters, 196, 334.
Akhtar Hayat is very grateful to the Higher Education Commission of Pakistan for financial support. This study was carried out as the part of the research project ALARMTOX, INTERREG SUDOE IVB and FEDER through the SOE1/P1/E129.
About this article
Cite this article
Hayat, A., Barthelmebs, L. & Marty, JL. A Simple Colorimetric Enzymatic-Assay for Okadaic Acid Detection Based on the Immobilization of Protein Phosphatase 2A in Sol-Gel. Appl Biochem Biotechnol 166, 47–56 (2012). https://doi.org/10.1007/s12010-011-9402-0
- Okadaic acid
- Protein phosphatase 2A
- Sol gel
- Storage test
- Colorimetric enzymatic-assay