Development of a handheld PEST-pen device for the rapid detection of organophosphate insecticides on food and clothing

摘要 有机磷（OP）杀虫剂对人体的神经毒性可通过皮肤或 吸入暴露于衣服和机器或口服暴露于食物残留物。在后一 在这方面，与使用OP杀虫剂有关的健康后果，特别是在 儿童，可以减少监测生产和消除消费的OP- 被污染的食物PlantVax目前雇用本氏烟草（Nicotiana benthamiana（N.b.）植物 表达系统以产生高水平的重组人乙酰胆碱酯酶（rHuAChE） 用于FLIR CAD笔等设备中，用于检测痕量OP神经 用于军事目的。在这项提案中，PlantVax旨在为食品安全目的开发， 一种简单的、手持式、便携式、独立的和廉价的PEST笔， 硫代磷酸酯杀虫剂在食品、衣物和机械上的应用。然而，与活动OP神经不同， OP杀虫剂表现出宽范围的双分子速率常数（ki），范围为ki = 101 M-1 min-1至109 M-1 min-1）对酯酶如AChE的抑制作用。这分为两类：（一） 具有高Ki（> 104 M-1 min-1），含有“oxon样”键，可立即用于 在阶段II中产生功能性PEST-笔和（ii）具有低ki（~101- 3 M-1 min-1）的那些，其具有 P=S键，需要通过氧化脱硫转化为活性氧磷形式， 细胞色素P450和NADPH的毒性和检测。新的PEST笔设备将 使用与CAD笔相同的基于酯酶的嵌入式化学物质，颜色为黄色至红色 改变为读出，但需要两个重要的修改，以增加ki， 颜色变化（i）使用也在N.b.中产生的重组人CES（rHuCES）。植物 固定到笔海绵中作为靶酶，因为I期结果表明， 1，000倍增加ki对硫和oxon形式与这种酶和（ii） 将P450和NADPH整合到笔中，将kilow转换为kihigh，以检测 许多高毒性有机磷杀虫剂，否则可能会被发现。在第二阶段， 化学将在20支功能性打印笔中进行优化，然后是2000支beta- 与化学相容的原型PEST笔将由以下公司设计和生产： 用于现场测试的注塑成型。计划初步测试蔬菜和水果样本， 市场和农场在美国，亚洲和中美洲，以证明实用的PEST- 笔全球。

ABSTRACT The neurotoxicity of organophosphate (OP) insecticides in humans may result from dermal or inhalation exposure on clothing and machinery or orally from the residue on food. In the latter context, the health consequences associated with the use of OP insecticides, especially in children, could be reduced by monitoring produce and eliminating the consumption of OP- contaminated food. PlantVax currently employs the Nicotiana benthamiana (N.b.) plant expression system to produce high levels of recombinant human acetylcholinesterase (rHuAChE) which is used in devices e.g. the FLIR CAD-pen, for the detection of trace amounts of OP nerve agents for military purposes. In this proposal, PlantVax aims to develop for food safety purposes, a simplified, hand-held, portable, self-contained and inexpensive PEST-pen to rapidly detect phosphorothionate insecticides on food, clothing and machinery. However unlike active OP nerve agents, OP insecticides exhibit a wide range of bimolecular rate constants (ki) ranging from ki = 101M-1min-1 to 109M-1min-1) against esterases e.g AChE. These fall into two categories (i) those with high ki (>104M-1min-1) which contain an “oxon-like” bond and can be used immediately to produce a functional PEST-pen in Phase II and (ii) those with low ki (~101-3M-1min-1) which have a P=S bond and will need to be converted by oxidative desulfuration into the active oxon form by cytochrome P450 and NADPH for their toxicity and detection. The new PEST-pen device will utilize the same esterase-based embedded chemistry as the CAD-pen with a yellow to red color change as a read-out, but with two important modifications required to increase the ki and thus the color change (i) the use of recombinant human CES (rHuCES) also produced in N.b. plants immobilized into the pen sponge as the target enzyme since Phase I results indicated a 10-fold to 1,000-fold increase in ki against both thion and oxon forms with this enzyme and (ii) the integration of P450 and NADPH into the pen to convert kilow to kihigh to enable the detection of many highly toxic OP insecticides which might have otherwise gone undetected. In Phase II, the chemistry will be optimized in twenty functional printed pens and then two thousand beta- prototype PEST-pens, which are compatible with the chemistry, will be designed and produced by injection molding for testing in the field. It is planned to initially test vegetable and fruit samples in markets and farms in the USA, Asia and Central America to demonstrate the utility of the PEST- pen globally.