基于溶胶-凝胶包埋的蛋白磷酸酶抑制比色法快速检测腹泻性贝类毒素

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

基于腹泻性贝类毒素(Diarrhetic shellfish poisoning,DSP)的主要成分大田软海绵酸(Okadaic acid,OA)及其衍生物对蛋白磷酸酶2A(Protein phosphatase 2A,PP2A)的抑制作用,采用改进的溶胶-凝胶(Sol-gel)技术包埋PP2A,建立了快速测定贝类中DSP的新方法。以对硝基苯磷酸二钠(4-Nitrophenyl phosphate disodium,p-NPP)为底物,利用碱性条件下PP2A催化p-NPP产生的黄色水解产物对硝基苯酚(4-Nitrophenol,p-NP)在405 nm处具有强烈吸收峰,而OA及其衍生物对PP2A的抑制会减少p-NP的生成,由此建立剂量-反应关系实现对DSP定量检测。本方法基于Sol-gel技术包埋PP2A,引入D-葡萄糖增大酶凝胶微孔的孔径,添加羟乙基纤维素防止酶凝胶发生脆裂,提高和保持酶的活性及稳定性,从而改善蛋白磷酸酶抑制比色法的重现性和可靠性。本方法的检出限为50μg OA eq./kg贝组织,线性范围为50~800μg/kg;加标回收率在68.8%~119.4%之间,相对标准偏差(RSD)为5.5%~14.8%。本方法将PP2A包埋固定于微孔板底部,可在-18℃条件下保持酶活性4个月以上,使用时直接加入底物和样品溶液进行反应,省去了传统PP2A抑制比色法中每次测试需重新配制酶溶液并进行水解等预处理步骤,使DSP的检测更简便、高效。

Based on the inhibition of protein phosphatase 2 A（ 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（ pNPP） 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.