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

开发手持式 PEST 笔装置，用于快速检测食品和衣物上的有机磷杀虫剂

• 批准号: 10079784
• 负责人: Yvonne J Rosenberg
• 金额: $ 70.58万
• 依托单位: PLANTVAX, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079784
• 关键词: 3D Print; Acetylcholinesterase; Ache; Adopted; Agricultural Crops; Asia; Blood; Brain; Categories; Central America; Cessation of life; Chemistry; Child; Chlorpyrifos; Cloning; Clothing; Color; Consumption; Country; Cytochrome P450; Data; Detection; Development; Devices; Diagnostic; Engineering; Environmental Health; Enzymes; Erythrocytes; Exhibits; Farming environment; Food; Food Contamination; Food Processing; Food Safety; Food production; Hand; Health; Human; Human Resources; Immobilization; In Vitro; Individual; Inhalation Exposure; Injections; Insecta; Insecticides; Kinetics; Military Personnel; Modification; Molds; Monitor; Multienzyme Complexes; NADP; Nicotiana; Oral; Organophosphates; Paraoxon; Parathion; Pesticides; Phase; Plants; Poison; Porifera; Production; Property; Recombinant Proteins; Recombinants; Sampling; Scientist; Site; Small Business Innovation Research Grant; Surface; System; Technology; Testing; Toxic effect; Training; base; biomaterial compatibility; carboxylesterase; cost; dermal exposure; design; detector; esterase; experience; falls; field study; fruits and vegetables; inhibitor/antagonist; nerve agent; neurotoxicity; portability; prototype; rapid detection; response; tool; toxic organophosphate insecticide exposure

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.

摘要 有机磷(OP)杀虫剂对人体的神经毒性可能源于皮肤或 吸入暴露在衣物和机器上或口服食物上的残留物。在后者中 在此背景下，与使用有机磷杀虫剂有关的健康后果，特别是在 可以通过监测产品和消除OP-2的消费来减少儿童死亡率。 受污染的食物。PlantVax目前雇用的是尼古丁·本特米亚纳(N.B.)种 高效表达重组人乙酰胆碱酯酶(RHuAChE)的表达系统 它用于例如FLIR CAD-PEN的设备中，用于检测微量的OP神经 用于军事目的的特工。在这项提案中，PlantVax旨在为食品安全目的开发， 一种简单、手持、便携、自给式、廉价的虫笔，可快速检测 食品、衣物和机械上的硫代磷酸盐杀虫剂。然而，与活跃的OP神经不同 杀虫剂、OP杀虫剂表现出很大范围的双分子速率常数(Ki)，范围从Ki= 101M-1min-1至109M-1min-1)抗酯酶，如AChE。这些可分为两类：(一)那些 具有高KI(&gt；104m-1min-1)，其包含“类氧”键，并且可立即用于 在第二阶段生产功能性害虫围栏，以及(II)低KI(~101-3M-1min-1)具有 P=S键，需要通过氧化脱硫法转化为活性氧子形式 细胞色素P450和NADPH的毒性及检测。新的虫笔装置将 使用与CAD笔相同的基于酯酶的嵌入化学物质，颜色从黄色到红色 改变为读出，但需要两个重要的修改来增加Ki，从而 颜色的变化(I)使用重组人CES(RHuCES)也是在N.B.植物 固定在PEN海绵中作为目标酶，因为I相结果显示10倍于 使用这种酶，KI对Thion和Oxon形式的KI增加1,000倍，以及(Ii) 将P450和NADPH整合到笔中，将KOW转换为KHIGH，使检测成为可能 许多剧毒的有机磷杀虫剂，否则可能不会被发现。在第二阶段， 化学将在20个功能打印笔上进行优化，然后是2000个测试版 与化学物质兼容的害虫笔原型将由 用于现场测试的注塑成型。计划首先测试蔬菜和水果样本。 在美国、亚洲和中美洲的市场和农场展示这种害虫的效用- 笔全球。