Citation: CHEN Jia-Qi, WU Hai-Yan, YAO Lin, ZHENG Guan-Chao, GUO Meng-Meng, TAN Zhi-Jun, ZHAI Yu-Xiu, MU Hai-Jin. Rapid Determination of Diarrhetic Shellfish Poisoning in Shellfish by Colorimetric Protein Phosphatase Inhibition Assay Based on Immobilization of Protein Phosphatase 2A in Sol-Gel[J]. Chinese Journal of Analytical Chemistry, ;2018, 46(8): 1261-1268. doi: 10.11895/j.issn.0253-3820.171529 shu

Rapid Determination of Diarrhetic Shellfish Poisoning in Shellfish by Colorimetric Protein Phosphatase Inhibition Assay Based on Immobilization of Protein Phosphatase 2A in Sol-Gel

1.
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;
2.
Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture, P. R. China;Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

Received Date: 26 December 2017
Revised Date: 6 June 2018

Fund Project: This work was supported by the Key Research Project of Shandong Province (2016GSF120018), the National Natural Science Foundation of China (No. 31772075) and Central Public-interest Scientific Institution Basal Research Fund, CAFS (No. 2016HY-ZD1101,2017HY-YJ0201).

Based on the inhibition of protein phosphatase 2A (PP2A) by diarrhetic shellfish poisoning (DSP), mainly okadaic acid (OA) and its analogues (dinophysistoxins, DTXs), colorimetric protein phosphatase inhibition assay (PPIA) was built to detect DSP in shellfish in which 4-nitrophenyl phosphate (p-NPP) was used as the substrate. After hydrolyzed by PP2A, the product (4-nitrophenol, p-NP) was measured at 405 nm, then toxins in samples could be quantified according to the standard dose-effect curve developed with a series of toxin standard solutions. In order to overcome the instability of PP2A and preserve the enzymatic activity well, sol-gel, accompanied with hydroxyethyl-cellulose (HEC), was used to encapsulate and immobilize PP2A at the bottom of the microwells. Also, D-glucose was employed as the template or pore-forming agent to improve the sensitivity. The PP2A immobilized sol-gel exhibited a storage stability of 4 months at -18℃. The limit of detection was 50 μg OA eq./kg, the spiked recoveries for OA in shellfish samples were between 68.8% and 119.4%, and the linear range was from 50 to 800 μg/kg. The PPIA with good recovery and reproducibility was simple and convenient. Samples and substrate solutions were added directly when used, demonstrating this proposed method could be used as an efficient analysis tool for rapid screening of DSP and be suited for regular monitoring program to control shellfish toxicity.
- Diarrhetic shellfish poisoning,
- Colorimetric protein phosphatase inhibition assay,
- Sol-gel,
- Shellfish,
- Rapid detection
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沈阳化工大学材料科学与工程学院沈阳 110142