SBIR-STTR Award

The neurotoxicity of OP pesticides in humans may result from dermal or inhalation exposure on clothing or machinery or orally from the residue on food. In the latter context, the health consequences associated with the use of organophosphate (OP) pesticides, especially in children, could be reduced by monitoring produce and eliminating the consumption of OP-contaminated food. PlantVax currently employs the Nicotiana benthamiana plant expression system to produce high levels of recombinant human acetylcholinesterase (rHuAChE) which is used in devices eg the FLIR CADpen, for the detection of trace amounts of organophosphate (OP) nerve agents for military purposes. In this proposal, PlantVax aims to develop a simplified, hand-held inexpensive PEST-pen to rapidly detect phosphorothionate insecticides on food, clothing and machinery. While the new device will utilize the same esterase-based embedded chemistry as the CADpen with a yellow to red color as a read-out, important features need to be altered to increase the rate of color change efficacy of the new device. Firstly, the inhibition constants (ki) obtained for carboxylases (CES) are higher for insecticides than HuAChE and will initially be incorporated into the pen sponge. Secondly since phosphorothionate insecticides are inactive and need to be metabolically converted by oxidative desulfuration into the active oxon form by cytochrome P450 in the liver for their insecticidal action, the PEST-pen may need to integrate features of both thion oxidation to an oxon as well as CES/AChE inhibition into a single device. The innovation in this proposal will be the identification of novel indicator molecules with high inhibition rates for incorporation into the sponge. These include (i) the wildtype or G137D mutant form of the carboxylesterase enzyme from the Australian blow fly Lucilia cuprina (Lc?E7) which exhibits high kinetic parameters for thion forms or (ii) mutant AChE enzyme with increased rates of inhibition by oxon embedded together with a P450, in the presence of a cytochrome c reductase (NADPH), to expedite conversion to the oxonforms. Based on the design of the successful CADpen, several PEST-pen prototypes with different indicator molecules will be produced and tested within a year.

Public Health Relevance Statement:


Project narrative:
While organophosphate (OP) insecticides greatly increase yields of agricultural crops, they can cause neurotoxicity and even death in humans as a result from dermal or inhalation exposure on clothing or orally from residue on food. In order to eliminate consumption of OP-contaminated food, PlantVax plans to develop a simple, hand-held, inexpensive device called a PEST-pen to monitor and rapidly detect OP insecticides on food, clothing and machinery.

Project Terms:
Acetylcholinesterase; Ache; Agricultural Crops; Agriculture; base; Blood; Blowfly; Brain; Buffers; Carbamates; carboxylesterase; Cessation of life; Chemistry; Child; Chlorpyrifos; Clothing; Color; Consumption; Cytochrome a; Cytochrome c Reductase; Cytochrome P450; dermal exposure; design; Detection; detector; Development; Device Designs; Devices; Diagnostic; Engineering; Environmental Health; Enzymes; Equilibrium; Erythrocytes; esterase; Exhibits; experience; Family; fly; Food; Food Contamination; Food Processing; Hand; Health; Human; In Situ; Individual; Inhalation Exposure; inhibitor/antagonist; innovation; Insecta; Insecticides; Kinetics; Liver; Malathion; Metabolic; Military Personnel; Monitor; Multienzyme Complexes; mutant; NADP; nerve agent; neurotoxicity; Nicotiana; non-Native; novel; Oral; Organophosphates; oxidation; Parathion; Pesticides; Phase; Plants; Poisons; Porifera; Production; prototype; rapid detection; Recombinants; response; Scientist; Signal Transduction; Site; Small Business Innovation Research Grant; Surface; System; Technology; Testing; Time; tool; Toxic effect; toxic organophosphate insecticide exposure

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.

Public Health Relevance Statement:


Project narrative:
While organophosphate (OP) insecticides greatly increase yields of agricultural crops, they can cause neurotoxicity and even death in humans as a result from dermal or inhalation exposure on clothing or orally from residue on food. In order to eliminate consumption of OP-contaminated food, PlantVax plans to develop a simple, hand-held, inexpensive device called a PEST-pen to monitor and rapidly detect OP insecticides on food, clothing and machinery.

Project Terms:
3D Print; Acetylcholinesterase; Ache; Adopted; Agricultural Crops; Asia; base; biomaterial compatibility; Blood; Brain; carboxylesterase; Categories; Central America; Cessation of life; Chemistry; Child; Chlorpyrifos; Cloning; Clothing; Color; Consumption; cost; Country; Cytochrome P450; Data; dermal exposure; design; Detection; detector; Development; Devices; Diagnostic; Engineering; Environmental Health; Enzymes; Erythrocytes; esterase; Exhibits; experience; falls; Farming environment; field study; Food; Food Contamination; Food Processing; Food production; Food Safety; fruits and vegetables; Hand; Health; Human; Human Resources; Immobilization; In Vitro; Individual; Inhalation Exposure; inhibitor/antagonist; Injections; Insecta; Insecticides; Kinetics; Military Personnel; Modification; Molds; Monitor; Multienzyme Complexes; NADP; nerve agent; neurotoxicity; Nicotiana; Oral; Organophosphates; Paraoxon; Parathion; Pesticides; Phase; Plants; Poison; Porifera; portability; Production; Property; prototype; rapid detection; Recombinant Proteins; Recombinants; response; Sampling; Scientist; Site; Small Business Innovation Research Grant; Surface; System; Technology; Testing; tool; Toxic effect; toxic organophosphate insecticide exposure; Training