Device and method for detecting organophosphates

Abstract

Device and method for detecting organophosphates. The present invention relates to a device that can be used to detect organophosphates and carbamates on surfaces including food, clothing (including as wearable pesticide detectors) and machinery.

Description

1 / 68 DEVICE AND METHOD FOR DETECTING ORGANOPHOSPHATES

[001] The present invention was made with the support of the Government under grant NIH No. 1R43ES029405. The Government may have certain rights in the present invention. Field of Invention

[002] The present invention relates to a device that can be used to detect organophosphate (OP) and carbamate (C) compounds on surfaces including food, clothing (including as wearable pesticide detectors), environmental samples and machinery. Description of the Related Technique

[003] Worldwide, the routine use of pesticides, including organophosphates, to control agricultural, domestic, and structural pests has reached more than 5 billion tons annually, potentially exposing more than 1.8 billion civilians and tons of agricultural products. In the US, levels are high enough to result in 10,000 to 20,000 pesticide poisonings among just ~2 million agricultural workers annually. Although pesticides greatly increase food production by reducing insect infestations, they are toxic compounds and have environmental and health effects.WHO Class I and Class II OP and carbamate pesticides constitute a diverse group of chemical structures, but all potentially exhibit a common mechanism of toxicity similar to nerve agents, namely, modification of the acetylcholinesterase (AChE) active site resulting in its inhibition, accumulation of acetylcholine, overstimulation of cholinergic receptors and consequent clinical signs of cholinergic toxicity.

[004] Although safe for humans and other mammals at the low doses used, there is growing concern about the effects of long-term exposure to these chemicals by agricultural workers and the level of Petition 870210102169, dated 05 / 11 / 2021, pages 264 / 341 2 / 68 of pesticides are consumed with food. This is especially true in Asia. At high exposures, acute toxicity can occur leading to seizures, brain damage, and cognitive and behavioral deficits, and often death from respiratory failure. In addition to occupational exposure to prolonged or high doses of pesticides, OPs and carbamates, their potency has been associated with a major cause of disability and death. In this context, insecticide poisoning is often the preferred method of suicide in Asia, killing more than 100,000 people annually in India alone. Furthermore, pesticide use has been associated with neurocognitive deficits and neuroendocrine changes described in veterans as Gulf War syndrome, and more recently, pesticides are considered to have been used by Islamic terrorists to attack schools in Afghanistan from 2010 to 2013, harming more than 2,000 girls.

[005] Neurotoxicity caused by insecticide spraying can result from dermal exposure or inhalation of particles in the air, on clothing or machinery, or orally from residues in food. In the latter context, the effect on children appears to occur at lower levels than for adult exposure. These health consequences, particularly associated with the use of OP and carbamate (C) insecticides, could be reduced by monitoring the production and eliminating the consumption of OP-contaminated foods. See, for example, www.who.int / ipcs / publications / pesticides_hazard_2009.pdf. In the US, the permitted pesticide residue level in the food we eat will likely be determined by decisions made based on specific pesticide use and environmental and health assessments.In Asia, however, monitoring insecticides in food and health concerns may be important factors in decisions about pesticide use, particularly for exported crops.

[006] Although the US EPA has banned most residential uses Petition 870210102169, dated 05 / 11 / 2021, pages 265 / 341 Although the reduction in the use of organophosphates (OPs) in 2001, as well as some for agricultural purposes, has resulted in decreases in both the level and percentage of OP insecticides used in the U.S., approximately 20 million pounds of OP pesticides were still sprayed agriculturally on fruits and vegetables in 2012; representing 33% of all insecticides (EPA Pesticide Industry Sales and Usage 2008-2012 estimates). The most widely used OP, chlorpyrifos, although now under pressure, still ranks as the fourteenth most commonly used conventional pesticide in the U.S. and has recently been linked to autism and ADHD (EPA Revised Human Health Risk Assessment for Registration Review, Nov 2016). Aldicarb, the active substance in the pesticide Temik, is one of the most widely used insecticides and also one of the most environmentally toxic.One consideration for banning all OPs is that, unlike other pesticides, they are slowly hydrolyzed in a humid atmosphere and in water, and show a low propensity to move up the food chain as happened with DDT and other chlorohydrocarbons. However, unlike the US, Australia, and the European Union, which have banned or severely restricted many pesticides, their use in Asia and developing countries is still prevalent, and even parathion is still widely used despite its ban. Thus, a very large market will exist domestically and abroad for many years exclusively for OP / C pesticides. Furthermore, monitoring the use and residues of other pesticides will become increasingly important due to growing awareness and concerns about environmental and health impacts.Recent reports from Germany have shown a significant decline in insect populations and diversity, and this is becoming a major public concern regarding the use of insecticides in agriculture. This anticipates a step towards increasing the demand for rapid testing of environmental samples for pesticides, including organophosphates. Petition 870210102169, dated 05 / 11 / 2021, pp. 266 / 341 4 / 68

[007] Several biosensor devices have been developed for the detection of pesticides, OP insecticides, and nerve agents based on electrical, amperometric, spectroscopic, and color readings. For example, available pesticide detection kits include the NIDS Rapid Pesticide Test kit (ANP Technologies), Pesticide Detection cards (RenekaBio), and the Agri-Screen Ticket kit (Neogen). However, these kits are multi-component, have imprecise outcomes, require long incubation periods, and / or require cutting food or testing fluids after washing. Thus, the use of these kits at testing sites to obtain rapid results (in less than 20 minutes) or to test more than 1,000 fruits / vegetables within a few hours (e.g., 1 to 8 hours), for example, is not practical or even possible, and high-throughput economic screening of agricultural products for consumer safety and assurance is therefore not feasible.However, rapid on-site testing is an essential prerequisite for removing contaminated food from the market to effectively protect consumers and detect illegal use.

[008] Because the kits currently on the market for detecting insecticides and other pesticides have shown to have inaccurate outcomes, require long incubation periods and use complex kits with multiple solutions, what is needed is a more robust and independent test that detects OP / C rapidly (e.g., 2 to 20 minutes) and with high sensitivity and selectivity. Summary of the Invention

[009] This summary is provided to introduce a selection of concepts in a simplified form which are further described below in the Detailed Description. This summary is not intended to identify the key features or essential characteristics of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Petition 870210102169, dated 05 / 11 / 2021, pp. 267 / 341 5 / 68

[010] As described in the present invention, the innovative features are the engineering of the first efficient, small, inexpensive, portable device for the rapid, sensitive and specific detection of organophosphate and carbamate compounds on surfaces, agricultural products and environmental samples, without the need for sophisticated equipment.

[011] As described in the present invention, the present invention relates to a device for detecting an OP / C compound comprising the following elements: (1) a top piece comprising a first carrier material, wherein said first carrier material comprises an immobilized OP / C detection enzyme; (2) a first substrate; (3) a second enzyme; (4) a second substrate; (5) a pH-sensitive dye; (6) a second carrier material; (7) an ampoule comprising a buffer;(8) an intermediate piece and (9) a bottom piece, wherein the intermediate piece is associated with the top piece and the bottom piece, wherein the intermediate piece comprises the second carrier material and the ampoule and wherein when the intermediate piece is rotated relative to the top piece or the bottom piece, the ampoule is capable of being broken to release the stopper to contact the first carrier material and the second carrier material causing (i) the enzymatic conversion of the first substrate by the OP sensing enzyme to produce an acidic reaction product; and (ii) the enzymatic conversion of the second substrate by the second enzyme to produce a basic reaction product. This device may also include an oxidant.

[012] In preferred embodiments, the OP / C detection enzyme is (a) a hydrolase; (b) a lipase, a phosphatase, an amylase, a cellulase, a protease, a peptidase, a urease, or a deaminase; (c) a carboxylesterase (CES), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), organophosphorus hydrolase, or organophosphorus acid anhydrolase; (d) CES1 or Petition 870210102169, dated 05 / 11 / 2021, pages 268 / 341 6 / 68 CES2; (d) selected from Tables 2 to 5; or (e) a variant of the OP / C detection enzyme having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of the OP / C detection enzyme from (a) to (d). As described in the present invention, the variant of the OP / C detection enzyme either (1) retains the ability to hydrolyze the first substrate; or (2) retains that ability while being inhibited by an OP / C.

[013] In preferred embodiments, the OP / C detection enzyme: (a) can detect at least 10pg, at least 20pg, at least 30pg, at least 40pg, at least 50pg, at least 60pg, at least 70pg, at least 80pg, at least 90pg or at least 100pg of an OP / C compound; (b) can detect between 10 and 100pg, between 20 and 100pg, between 30 and 100pg, between 40 and 100pg, between 50 and 100pg, between 60 and 100pg, between 70 and 100pg, between 80 and 100pg, between 90 and 100pg of an OP / C compound; (c) comprises an inhibition rate constant kide of at least 103Mimin'1 to 108M'1-min'1, at least 104Mimin'1 to 108M'1^min'1, at least 105Mimin'1 to 108M'1-min'1, at least 106Mimin'1 to 108M'1^min'1 or at least 107M'1-min'1 to 108M'1-min'1; and / or (d) comprises an inhibition rate constant ki of 103 to 105Mimin'1, ki of 104 to 105M' ^min'1, 105 to 106Mimin'1, 106M -min1 to 107Mimin'1 or 106Mimin'1 to 108Mimin'1.

[014] In preferred embodiments, the first carrier material comprises: (a) natural polymers, including but not limited to cellulose, hemicellulose, pectin, chitin, silk, lignin, starch, polypeptides, collagens, keratins, polysaccharides, nucleic acids and / or rubbers; or (b) derivatives of natural polymers, including but not limited to methylation, Petition 870210102169, dated 05 / 11 / 2021, pages 269 / 341 7 / 68 carboxylation, amidation, sulfation, hydroxylation, condensation, iodination, reduction, oxidation, esterification, alkylation and / or halogenation; and / or (c) synthetic polymers and copolymers, including, but not limited to, polyurethanes, thermoplastic polyurethanes, silicones, polyamides, polystyrenes, bakelite, polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polychloroprene and / or polyimides. In preferred embodiments, the first carrier material is a sponge and / or is made of polyurethane.

[015] In additional embodiments, the first substrate is selected from acetylcholine, butyrylcholine-4-nitrophenyl acetate, 4-nitrophenyl propionate, 4-nitrophenyl butyrate, 4-nitrophenyl valerate, 4-nitrophenyl dimethylacetate, 4-nitrophenyl trimethylacetate, 4-nitrophenyl 4-guanidinobenzoate, n-glycero-3-phosphocholine or 6-nitrocoumarin. The first substrate may also be selected from thioesters such as acetylthiocholine, butyrylthiocholine, S-4-nitrobenzyl thioacetate, S-phenyl thioacetate.

[016] In additional embodiments, the second enzyme and the second substrate are selected from Table 6. In additional preferred embodiments, the second enzyme is urease, the second substrate is urea, and / or the basic reaction product is ammonia.

[017] In additional preferred embodiments, the pH-sensitive dye is selected from nitrazine, phenol red, chlorophenol red, bromocresol green, cresol red, bromomethyl blue or bromocresol violet.

[018] In certain embodiments, the oxidant is included in the device and converts an inactive OP / C compound to an active OP / C compound. Examples of such oxidants include, but are not limited to, Fenton, a halogen (e.g., iodine, bromine, chlorine, and fluorine), or a P450 enzyme in the presence of the cofactor NADPH. The preferred example of the P450 enzyme is the wild-type mutant or Petition 870210102169, dated 05 / 11 / 2021, pages 270 / 341 8 / 68 triple CYP1A2 (P450 BM-3 (CYP102-A1).

[019] In preferred embodiments, in addition to the OP / C detection enzyme, the first carrier material may also comprise the first enzyme, the second enzyme and / or the oxidant. In other embodiments, the ampoule also comprises the pH-sensitive dye; and / or the second carrier material comprises the pH-sensitive dye, the first substrate, the second substrate and / or the oxidant.

[020] In additional preferred embodiments, the second carrier material is selected from: a test strip comprising dry filter paper or a second polymer.

[021] In additional preferred embodiments, the pH-sensitive dye, the first substrate, the second substrate and / or the oxidant are lyophilized as a microtablet.

[022] In further preferred embodiments, the top piece and the middle piece are connected. Additionally, in preferred embodiments the ampoule extends into the bottom piece. In further embodiments, the middle piece contains one or more holes to allow the flow of contents released from the ampoule between the bottom piece and the middle piece. Additionally, the device, as described in the present invention, further comprises a lid and this lid may be transparent and / or comprise a window.

[023] In additional preferred embodiments, the device comprises at least one O-ring which can be placed between the top piece and the intermediate piece and / or between the intermediate piece and the bottom piece to ensure the sealing of the connected pieces so that the reaction solutions remain in place.

[024] In additional preferred modes, the device is associated Petition 870210102169, dated 05 / 11 / 2021, pp. 271 / 341 9 / 68 operationally to a smartphone.

[025] As described in the present invention, additional embodiments include a method for detecting an OP / C comprising: (a) contacting the device, as described in the present invention, with a surface; (b) rotating the intermediate piece relative to the top piece or bottom piece thereby breaking the ampoule to release the buffer to contact the first carrier material and the second carrier material causing enzymatic conversion of a second substrate by a second enzyme; and wherein: (1) in the absence of an OP / C, enzymatic conversion of the first substrate by the OP / C detection enzyme occurs, resulting in maintenance of a reference pH; or (2) in the presence of an OP / C, enzymatic conversion of the first substrate by the OP / C detection enzyme is inhibited by the OP / C compound, resulting in an increase in pH above the reference pH due to the production of the basic reaction product.

[026] As described in the present invention and as known in the art, many OPs and carbamates can be detected using the device or method. Specifically, the OP / C compound that can be detected includes, but is not limited to: (a) an insecticide selected from: acephate, aldicarb (Temik), carbachol, carbamate, carbaryl (Sevin), carbofuran (Furadan), carisoprodol, chlorfenvinphos, Chlorpyrifos-oxon, Chlorpyrifos, Dementon-S, Diazoxon, diazinon, Dichlorvos, tofosophosdicrotophos, dimethoate, dithiocarbamates, EA-3990, eserine, ethienocarb, etoprophos, ethylcarbamate, felbamate, fenobucarb, fenamiphos, ofosisocarbophos, Malathion, mebutamate, meprobamate, Metamidaphos, methomyl, methylcarbamate, methyl parathion, MethylPOX, monocrotophosophos, naled, neostigmine, omethoate, oxamyl, Paraoxon, Parathion, phorate, phosmet, phosphamidon, rivastigmine, T-1123, terbufos, tetrachlorvinphos, Tetriso, thiocarbamates (e.g., O-thiocarbamate or S Petition 870210102169, dated 05 / 11 / 2021, pp. 272 / 341 10 / 68 thiocarbamates), triazophosofos and / or tibamate; (b) a G agent, such as Tabun (GA), Sarin (GB), Clorsarin (GC), Soman (GD), methylsarin, n-butylsarin, isobutylsarin, n-propylsarin, ethylsarin (GE) and / or cyclosarin (GF), GV; (c) a V agent, such as EA-3148, VE, VG, VM, VP, VR, VS and / or VX; and / or (d) a Novichok officer, such as A-234.

[027] As also described in the present invention and in preferred embodiments, the device (a) can detect at least 10pg, at least 20pg, at least 30pg, at least 40pg, at least 50pg, at least 60pg, at least 70pg, at least 80pg, at least 90pg or at least 100pg of an OP / C compound; and / or (b) can detect between 10 and 100pg, between 20 and 100pg, between 30 and 100pg, between 40 and 100pg, between 50 and 100pg, between 60 and 100pg, between 70 and 100pg, between 80 and 100pg, between 90 and 100pg of an OP / C compound.

[028] Examples of surfaces that can be tested with the device, as described in this invention, include, but are not limited to, food, clothing or machinery.

[029] It should be understood that both the preceding general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the present invention as claimed. The accompanying drawings are included to provide a further understanding of the present invention and are incorporated into and form part of this descriptive report, illustrating various embodiments of the present invention and, together with the description, serve to explain the principles of the present invention. DESCRIPTION OF THE DRAWINGS

[030] The modalities are illustrated by way of example (and not limitation) in the figures of the attached drawings, in which similar references indicate similar elements and in which:

[031] Figure 1A shows the bi-molecular rate constants (ki) of extracts Petition 870210102169, dated 05 / 11 / 2021, pp. 273 / 341 11 / 68 of rHuCES (recombinant human carboxylesterase) derived from plants tested against a battery of OP insecticides.

[032] Figure 1B shows the in vitro conversion of parathion to paraoxon using cytochrome P450 (CYP1A2) / NADPH microsomes (Fisher Scientific).

[033] Figure 1C shows the bi-molecular rate constants of plant-derived rHuCE extracts against a battery of OP insecticides compared to purified rHuCE controls produced in E. coli.

[034] Figure 1D shows the results of a second experiment demonstrating the in vitro conversion of parathion to paraoxon using cytochrome P450 (CYP1A2) / NADPH microsomes. Inhibition rate constants (ki) increased against rHuCES of parathion (L) and chlorpyrifos (R) after conversion of their oxons following a 10 min incubation with NADPH and P450 (CYP) from two sources. Paraoxon was used as a control.

[035] Figure 2 shows a representative example of the OP / C detection enzyme sequences that can be used in the device as described in the present invention.

[036] Figure 3 shows the different individual components of the device.

[037] Figure 4 shows a top view of the device.

[038] Figure 5 shows the side view of the device.

[039] Figure 6 shows a close-up view of the device.

[040] Figure 7 shows an additional schematic representation of the device.

[041] Figure 8 shows the structures of the most commonly used OP insecticides, which show the presence of P=O and P=S linkages that determine their bi-molecular rate constants and toxicity against rHuCES. It should be noted that, currently, omethoate is the only exception because it has a Petition 870210102169, dated 05 / 11 / 2021, pp. 274 / 341 12 / 68 P=O bond and a low ki (101M-1^min-1) against CES possibly related to the leaving group slowing down the reaction or a steric hindrance effect. A carbamate is included since it also inhibits AChE and CES. DETAILED DESCRIPTION OF THE INVENTION

[042] Before describing the present invention in detail, it should be understood that the present invention is not limited to the materials or process parameters particularly exemplified, as these may certainly vary. It should also be understood that the terminology used in the present invention is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting to the use of alternative terminology to describe the present invention.

[043] All publications, patents and patent applications cited in the present invention, whether above or below, are hereby incorporated by reference in their entirety for all purposes. A. Definitions

[044] As used in this descriptive report and the accompanying claims, the singular forms “a”, “an” and “the” include plural nouns unless the content clearly dictates otherwise. For example, reference to “a polypeptide” includes a mixture of two or more such polypeptide molecules or a plurality of such polypeptide molecules. Similarly, reference to a “polynucleotide” includes a mixture of two or more such polynucleotide molecules or a plurality of such polynucleotide molecules.

[045] As used in the present invention, the term “comprise” or variations thereof such as “comprises” or “comprising” should be read to indicate the inclusion of any reported integer (e.g., an aspect, element, feature, property, method / process step) Petition 870210102169, dated 05 / 11 / 2021, pp. 275 / 341 13 / 68 or limitation) or group of integers (e.g., aspects, elements, characteristics, properties, method / process steps, or limitations) but not excluding any other integer or group of integers. Thus, as used in the present invention, the term comprising is inclusive and does not exclude additional, unreported integers or method / process steps.

[046] In embodiments of any of the compositions and methods provided in the present invention, comprising may be replaced with essentially consisting of or consisting of. The phrase essentially consisting of is used in the present invention to require the specific integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used in the present invention, the term consisting is used to indicate the presence of the reported integer (e.g., an aspect, element, feature, property, method / process step, or limitation) or group of integers (e.g., aspects, elements, features, properties, method / process steps, or limitations) alone.

[047] As used in the present invention, OP / C is used to define an organophosphorus or carbamate insecticide or nerve agent. Representative examples of OP / Cs include, but are not limited to: (a) an insecticide selected from: acephate, aldicarb (Temik, AgLogic 15G), carbachol, carbamate, carbaryl (Sevin), carbofuran (Furadan), carisoprodol, chlorfenvinphos, chlorpyrifos-oxon, chlorpyrifos, Dementon-S, Diazoxon, diazinon, Dichlorvos, tofosophosdicrotophos, dimethoate, dithiocarbamates, EA-3990, eserine, ethienocarb, etoprophos, ethylcarbamate, felbamate, fenobucarb, fenamiphos, ofosisocarbophos, Malathion, mebutamate, meprobamate, Metamidaphos, methomyl, methylcarbamate, methyl parathion, Methyl-POX, monocrotophosophos, naled, Petition 870210102169, dated 05 / 11 / 2021, pp. 276 / 341 14 / 68 neostigmine, omethoate, oxamyl, paraoxon, parathion, phorate, phosmet, phosphamidon, rivastigmine, T-1123, terbufos, tetrachlorvinphos, tetriso, thiocarbamates (e.g., O-thiocarbamate or S-thiocarbamate), triazophosphos and / or tibamate; (b) a G agent, such as Tabun (GA), Sarin (GB), Clorsarin (GC), Soman (GD), methylsarin, n-butylsarin, iso-butylsarin, n-propylsarin, ethylsarin (GE) and / or cyclosarin (GF), GV; (c) a V agent, such as EA-3148, VE, VG, VM, VP, VR, VS and / or VX; and / or (d) a Novichok officer, such as A-234.

[048] Since all OP / Cs function by inhibiting the ability of an OP / C-detecting enzyme to convert the first substrate, OP / C can be detected using the same colorimetric assay described in the present invention.

[049] Although not formally classified as OPs, the AChE inhibition mechanism also occurs with carbamate insecticides / nerve agents. Thus, the device described in the present invention can also be used for detected carbamate agents, including carbamate and / or carbamate insecticides / nerve agents. Examples of such agents include, but are not limited to: aldicarb (Temik), carbofuran (Furadan), carbaryl (Sevin), ethienocarb, fenobucarb, oxamyl, methomyl, T-1123, EA-3990, ethylcarbamate, methylcarbamate, neostigmine, rivastigmine, meprobamate, carisoprodol, felbamate, mebutamate, tibamate, carbachol, thiocarbamates (e.g., O-thiocarbamate, 5-thiocarbamates) and / or dithiocarbamates. As used in the present invention, OP or OPs will include carbamate insecticides / nerve agents.

[050] As used in the present invention, an OP / C detection enzyme refers to (a) a hydrolase; (b) a lipase, a phosphatase, an amylase, a cellulase, a protease, a peptidase, a urease or a deaminase; Petition 870210102169, dated 05 / 11 / 2021, pp. 277 / 341 15 / 68 (c) a carboxylesterase (CES), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), organophosphate hydrolase or organophosphate acid anhydrolase; (d) CES1 or CES2; (d) selected from Tables 2 to 5; or (e) a variant of the OP / C detection enzyme having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of the OP / C detection enzyme from (a) to (d). As described in the present invention, the OP / C detection enzyme variant both (1) retains the ability to convert the first substrate into acetic acid; and (2) retains this ability while being inhibited by an OP / C.Since the sequences for these enzyme families are known and published in public databases, they were not included in this descriptive report; however, they are incorporated herein by reference in their entirety if necessary. In particular, in preferred embodiments, the term “OP / C detection enzymes” also includes variants of such CES, AChE, or BChE enzymes such that the variant (a) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence from which it was derived, (b) retains the ability to convert the first substrate to acetic acid, and (c) retains this ability to be inhibited by an OP / C.Those skilled in the art will readily recognize that the method according to the present invention is not limited to any single enzymes or enzyme family and can generally be applied to enzymes that catalyze a first reaction leading to a decrease in pH. Thus, the device can be used for various reactions and enzymes including, but not limited to. Petition 870210102169, dated 05 / 11 / 2021, pp. 278 / 341 16 / 68 limited to hydrolases and oxidoreductases.

[051] In preferred embodiments, the OP / C detection enzyme: (a) can detect at least 10μg, at least 20μg, at least 30μg, at least 40μg, at least 50μg, at least 60μg, at least 70μg, at least 80μg, at least 90μg or at least 100μg of an OP / C compound; (b) can detect between 10 and 100μg, between 20 and 100μg, between 30 and 100μg, between 40 and 100μg, between 50 and 100μg, between 60 and 100μg, between 70 and 100μg, between 80 and 100μg, between 90 and 100μg of an OP / C compound; (c) comprises an inhibition rate constant ki of at least 103Mimin'1 to 108M'1-min'1, at least 104Mimin'1 to 108M' ^min'1, at least 105Mimin'1 to 108Mimin'1, at least 106Mimin'1 to 108M-1^min-1 or at least 107Mimin'1 to 108Mimin'1; and / or (d) comprises an inhibition rate constant ki of 103 to 105Mimin'1, ki of 104 to 105M'1-min'1, 105 to 106Mimin'1, 106Mimin'1 to 107Mimin'1 or 106Mimin'1 to 108Mimin'1.

[052] As used in the present invention, when an OP / C is classified as “not detected” by the device as described in the present invention, there may still be very low quantities of OP / C present on the surface. However, the quantity is at a level that is below the detection limit of the device.

[053] As used in the present invention, “reference pH” refers to the pH or change in pH in the absence of any inhibitor of the first enzyme, i.e., the OP / C detection enzyme. This reference pH is adjusted by the two reactions that occur within the device after the buffer is released from the ampoule. The conversion of the first substrate by the first enzyme acidifies, i.e., decreases the pH of the reaction buffer, and the conversion of the second substrate by the second enzyme basifies, i.e., increases the pH of the reaction buffer. The reaction rates for the first and second reactions are chosen such that the overall change in Petition 870210102169, dated 05 / 11 / 2021, pp. 279 / 341 17 / 68 The pH is zero (idealized) or decreases slightly, and the pH-responsive molecule, for example, the halochromic chemical compound (pH indicator), does not change its optical properties. However, in the presence of an inhibitor of the first enzyme, the reaction rate of the first reaction, and thus the acidification (decrease in pH) due to the conversion of the first substrate, is reduced, resulting in a net increase in pH evidenced by the color change of the pH indicator. The rate and degree of color change reflect the inhibition kinetics and the bimolecular rate constant (ki) of the first enzyme for OP / C and the amount of inhibitor present, i.e., OP / C pesticide for an OP / C detection enzyme. In preferred embodiments, the increase in pH is indicated when pH levels of at least 0.5, at least 1.0, at least 2.0, or at least 3.0 have been obtained. In preferred embodiments, a product of the second reaction is ammonia.

[054] As used in the present invention, a CES is an enzyme classified as a carboxylesterase, which is a well-studied multigene family of enzymes (EC 3.1.1.1) widely found in organisms ranging from bacteria to mammals. These enzymes are members of the serine hydrolase superfamily, in which a serine residue is involved in the hydrolysis of ester, amide, or carbamate linkages. See, for example, Sogorb MA, Vilanova E. Enzymes involved in the detoxification of organophosphorus, carbamate, and pyrethroid insecticides through hydrolysis, Toxicol. Lett. (2002) 128:215-228. Organophosphate, carbamate, and pyrethroid insecticides are metabolized by CES. The acyl-binding pocket of the OP / C binding site (Hopkins et al, Biochemistry (2017) 56:5512-5525). A recent genomic analysis defined five distinct subfamilies of mammalian CES (Williams et al.(2010) based on genetic sequence and genomic structure, with proteins from the CES1 and CES2 subfamily being the most extensively studied. Sequence similarities exist. Petition 870210102169, dated 05 / 11 / 2021, pages 280 / 341 18 / 68 significant for the five CES families, especially for key regions previously identified for human hepatic CES1 (Bencharit et al. 2003, 2006; Fleming et al. 2005). Three-dimensional structural analyses of human CES1 identified three main ligand-binding sites, including the broad-specificity active site, the “side gate,” and the “Z site,” where substrates, fatty acids, and cholesterol analogs, respectively, are bound; and an active site 'gate,' which may facilitate product release after catalysis (Bencharit et al. 2003, 2006; Fleming et al. 2005). The acyl-binding pocket of the OP / C binding site. See, for example, Holmes et al., Mamm. Genome. 2010 Oct; 21(9 to 10): 427-441 for further description of amino acid conservation among CES subfamilies, crystal structure and conserved amino acids among different CES species (incorporated into the present invention by reference in its entirety).As used in the present invention, any known CES enzymes (see, for example, Figure 2, Table 2 and / or the enzymes described in Holmes et al.) can be included in the device described in the present invention and used to detect OP, as well as variants of such known CES enzymes that retain carboxylesterase activity. In preferred embodiments, CES1 or CES2 enzymes (including variants) are used.

[055] As used in the present invention, “AChE” refers to the class of proteins referred to as acetylcholinesterase and “BChE” refers to the class of proteins referred to as butyrylcholinesterase (“BChE”) (classified as EC 3.1.1.7 and EC 3.1.1.8 respectively). The 3D structure of acetylcholinesterase has been determined and published (e.g., PMID: 1678899). This protein has a 3-layer alpha-beta-alpha sandwich fold common to members of the alpha / beta hydrolase family. Surprisingly, given the high turnover rate of acetylcholinesterase, the active site of these enzymes has been determined to be located at the bottom of a deep, narrow cleft, called the Petition 870210102169, dated 05 / 11 / 2021, pp. 281 / 341 19 / 68 throat of the active site. As used in the present invention, any known AChE / BChE enzyme can be included in the device described in the present invention and used to detect OP, as well as variants of such known AChE / BChE enzymes. Representative examples of such AChE / BChE enzymes are shown in Tables 2 to 5 and Figure 2.

[056] As used in the present invention, a “first substrate” is used to refer to a molecule that can be enzymatically converted into an acid by the first enzyme, for example, an OP / C detection enzyme. Representative examples of a first substrate include, but are not limited to, acetylcholine, acetylthiocholine, butyrylcholine, butyrylthiocholine, 4-nitrophenyl acetate, 4-nitrophenyl propionate, 4-nitrophenyl butyrate, 4-nitrophenyl valerate, 4-nitrophenyl dimethylacetate, 4-nitrophenyl trimethylacetate, 4-nitrophenyl 4-guanidinobenzoate, or 6-nitrocoumarin. See, for example, Williams et al., Drug Metabolism and Disposition, Vol. 39, No. 12 (2011) (incorporated by reference in its entirety).

[057] As used in the present invention, a “pH-sensitive dye” refers to an indicator composition that is capable of undergoing an observable change of state (e.g., a change in optical properties / color) as a result of reactions occurring within the device. Preferably, such a dye changes optical properties in a manner that is visible to the human eye. Examples of pH-sensitive dyes include, but are not limited to: nitrazine, phenol red, chlorophenol red, bromocresol green, cresol red, bromomethyl blue, or bromocresol violet. The degree of color change may be correlated to the amount of conversion of the first substrate. Therefore, the varying degree of color change indicates not only the presence of an OP / C, but also the inhibition kinetics and the amount of OP / C present. Petition 870210102169, dated 05 / 11 / 2021, pages 282 / 341 20 / 68

[058] As described in the present invention, the conversion of the second substrate by the second enzyme results in the basification of the reaction buffer (i.e., the action or process of making something more basic resulting in an increase in the pH of something). Representative examples of a second substrate and second enzyme include, but are not limited to, urea and urease (classified as EC 3.5.1.5), urea and urea amidolyase (classified as EC 6.3.4.6 and EC 3.5.1.54), biuret and biuret amidohydrolase (classified as EC 3.5.1.84), [beta-hydroxypyruvate + glycolaldehyde] and transketolase (classified as EC 2.2.1.1, with representative substrate examples being: D-fructose 6-phosphate, D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate or D-xylulose 5-phosphate), adenosine and adenosine deaminase (classified as EC 3.5.4.4), adenine and adenine deaminase (classified as EC 3.5.4.15), guanosine and guanosine deaminase (classified as EC 3.5.4.15), guanine and guanine deaminase (classified as EC 3.5.4.3), cytosine and cytosine deaminase (classified as EC 3.5.4.5).

[059] As used in the present invention, an “oxidant” is used to refer to a molecule capable of converting an inactive phosphorothionate “thion” or carbamate form of an OP / C into an active form (e.g., oxon) (see Fig. 8). Representative examples of an oxidant include, but are not limited to: Fenton, a halogen (e.g., iodine, bromine, chlorine, and fluorine), or a P450 enzyme in the presence of the cofactor NADPH. The preferred example of the P450 enzyme is a triple mutant of CYP1A2 (P450 BM-3 (CYP102-A1)).

[060] In the present invention, a “polynucleotide” refers to the polymeric phosphate ester form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNA molecules”) or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in single-stranded or single-stranded helix form. Petition 870210102169, dated 05 / 11 / 2021, pp. 283 / 341 21 / 68 double. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, the sequences can be described in the present invention according to the normal convention of providing only the sequence in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand having a sequence homologous to mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation.

[061] The terms “percentage (%) of sequence similarity”, “percentage (%) of sequence identity” and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of several publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetic Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.

[062] To determine the percentage of identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number Petition 870210102169, dated 05 / 11 / 2021, pages 284 / 341 22 / 68 identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are approximately, the same length. The percentage of identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating the percentage of sequence identity, typically exact matches are counted.

[063] Determining the percentage of identity between two sequences can be performed using a mathematical algorithm. A non-limiting example of a mathematical algorithm used for comparing two sequences is the Karlin and Altschul algorithm, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the sequences of the present invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein sequences of the present invention.To obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform a repeated search that detects distant relationships between molecules. See Altschul et al. (1997) above. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov / BLAST / on the World Wide Web.

[064] Another non-limiting example of a mathematical algorithm used Petition 870210102169, dated 05 / 11 / 2021, pages 285 / 341 23 / 68 for sequence comparison is the Myers and Miller algorithm, CABIOS 1988; 4: 1 1 - 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[065] In a preferred embodiment, the percentage of identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 1970, 48:444-453), which was incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available at accelrys.com on the WorldWideWeb), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of length of 1, 2, 3, 4, 5 or 6.A particularly preferred set of parameters (and the only one that can be used if the practitioner is uncertain about which parameters should be applied to determine whether a molecule is a sequence identity or homology limitation of the present invention) is the use of a Blossum 62 scoring matrix with a gap opening penalty of 12, a gap extension penalty of 4, and a reading frame shift gap penalty of 5.

[066] In the present invention, “isolated polypeptide” means that the polypeptide is separated from its environment and is present in sufficient quantity to permit its identification or use. Isolated polypeptides include recombinantly produced polypeptides. This means, for Petition 870210102169, dated 05 / 11 / 2021, pp. 286 / 341 24 / 68 For example, the polypeptide can be (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. Isolated proteins or polypeptides can be, but need not be, substantially pure. Because an isolated polypeptide can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide may comprise only a small percentage by weight of the preparation. The polypeptide is, however, isolated by having been separated from substances with which it may be associated in living systems, for example, isolated from other proteins. Any of the peptides or polypeptides provided in the present invention can be isolated. B. Device

[067] As described in the present invention, the device is designed to contain all sensory components in a self-enclosed system that is substantially simpler to manufacture and use compared to the ten or more components used for other pens currently on the market for detecting nerve agents. This innovation provides an efficient, small, inexpensive, portable device for the rapid, sensitive and specific detection of OP / C.

[068] As shown in Figure 3, the device described in the present invention comprises the following components: a first carrier material (100) that fits into a top piece (110). An ampoule (120) is contained and protected by an intermediate piece (130) that connects to a bottom piece (140). The ampoule may also optionally be inside the bottom piece. The device may also optionally comprise a separate cover or lid; alternatively, as shown in Figure 3, the top piece (110) may be manufactured to include a cover or lid. In addition to the ampoule (120), the intermediate piece (130) also houses a second material. Petition 870210102169, dated 05 / 11 / 2021, pp. 287 / 341 25 / 68 carrier (not shown).

[069] The substrates and enzymes used to detect OP / C can be configured differently within the device. For example, the substrates, enzymes, and pH-sensitive dye can be configured in the following different ways based on the intended use. For example, short-term storage might consider the enzymes, substrates, and / or dyes to be included in the ampoule. Conversely, long-term storage would have a preferred configuration where only the buffer would be included in the ampoule. In additional preferred modes, the corresponding substrates and enzymes should not be configured in the same location within the device. Table 1 First carrier material Ampoule Second carrier material 1st Enzyme (1) Buffer 2nd Enzyme 1st and 2nd substrates pH-dependent dye 1st Enzyme pH-dependent dye Buffer 2nd Enzyme 1st and 2nd substrates 1st Enzyme Buffer pH-dependent dye 2nd Enzyme 1st and 2nd substrates 1st and 2nd Enzymes Buffer 1st and 2nd substrates pH-dependent dye 1st and 2nd Enzymes pH-dependent dye Buffer 1st and 2nd substrates 1st and 2nd Enzymes pH-dependent dye Buffer 2nd substrate 1st substrate 1st and 2nd Enzymes pH-dependent dye Buffer 1st substrate 2nd substrate 1st and 2nd Enzymes pH-dependent dye Buffer 1st and 2nd substrates Null 1st Enzyme Buffer pH-dependent dye 1st and 2nd substrates 1st and 2a pH-dependent dye buffer enzymes 1st and 2nd substrates Petition 870210102169, dated 05 / 11 / 2021, pp. 288 / 341 26 / 68 First carrier material Ampoule Second carrier material 1st Enzyme Buffer 2nd substrate 1st substrate 2nd Enzyme pH-dependent dye 1st Enzyme pH-dependent dye Buffer 2nd substrate 1st substrate 2nd Enzyme 1st Enzyme Buffer 2nd substrate pH-dependent dye 1st substrate 2nd Enzyme 1st and 2nd Enzymes Buffer 2nd substrate 1st substrate pH-dependent dye 2nd Enzyme Buffer 1st Enzyme 1st and 2nd substrates pH-dependent dye 2nd Enzyme pH-dependent dye Buffer 1st Enzyme 1st and 2nd substrates 2nd Enzyme Buffer pH-dependent dye 1st Enzyme 1st and 2nd substrates 2nd Enzyme Buffer 2nd substrate 1st Enzyme 1st substrate pH-dependent dye 2nd Enzyme pH-dependent dye Buffer 2nd substrate 1st Enzyme 1st pH-dependent dye substrate Buffer 1a and 2a Enzyme 1a Enzyme Buffer 1o and 2o pH-dependent dye substrates 2a Enzyme (1) For example, OP / C detection enzyme

[070] Within the context of the present invention, the term buffer means a composition (any combination) of water + / - solutes (including salts, including but not limited to, NaCl, KCl, MgSO4, CaCl, NiCl2, CuCh), a pH buffering compound (including salts, including but not limited to, Tris, MES, HEPES, Phosphate, Citrate), a reducing or antioxidant agent (2-ME, DTT, Na2S2O5, ascorbic acid, glutathione, cystine), an excipient (glucose, sucrose, glycerol, mannitol, proline, arginine, trehalose, erythritol, imidazole), a detergent (Tween-20, Tween-80, Triton-X100, Triton-X114, deoxycholic acid, maltoside, Petition 870210102169, dated 05 / 11 / 2021, pages 289 / 341 27 / 68 octyl-thioglucoside, CHAPS), a stabilizer (polyvinylpyrrolidone, chitosan, gelatin, elastin-like peptides, PEG, dendrimers, serum albumin, radical decontaminators, butylated hydroxytoluene, alkylated diphenylamine), preservative (benzoic acid, sulfur dioxide, gallic acid) or metal ion chelators (EDTA, EGTA).

[071] For example, the substrates and colorimetric reporters are dried on the second carrier housed in the intermediate piece (130), along with a glass ampoule (120) filled with diluted buffer. The enzymes (the OP / C detection enzyme and / or the second enzyme) can be immobilized covalently or non-covalently on the first carrier material (100).The carrier materials (the first and / or second carrier material) may be a natural polymer, including but not limited to cellulose, hemicellulose, pectin, chitin, silk, lignin, starch, polypeptides, collagens, keratins, polysaccharides, nucleic acids and / or rubbers; or (b) derivatives of natural polymers, including but not limited to methylation, carboxylation, amidation, sulfation, hydroxylation, condensation, iodination, reduction, oxidation, esterification, alkylation and / or halogenation; and / or (c) synthetic polymers and copolymers, including, but not limited to, polyurethanes, thermoplastic polyurethanes, silicones, polyamides, polystyrenes, bakelite, polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polychloroprene and / or polyimides) separated by the top piece (110) at the top of the device.In one embodiment, the OP / C detection enzyme and, optionally, the pH-dependent dye are incorporated into the polymer matrix of the first carrier material during polymer synthesis. For example, if polyurethane is used, the first carrier material can be formed by mixing water, the OP / C detection enzyme, optionally the pH-dependent dye, and an isocyanate-functionalized polyurethane prepolymer incorporating the OP / C detection enzyme, optionally the dye. Petition 870210102169, dated 05 / 11 / 2021, pp. 290 / 341 28 / 68 pH-dependent dye in the polymer network. See, for example, US 6,291,200 (incorporated by reference in its entirety).

[072] In a further embodiment, the second carrier material may comprise lyophilized substrate(s) and enzyme(s), for example, in the form of a powder, film or tablet. In another embodiment, the substrate(s), pH-dependent dye and / or enzyme(s) may be spatially separated in the second carrier material, for example, by drying the components in separate pieces. In yet another embodiment, the second carrier material may comprise two or more materials, for example, two different filter papers or a filter paper and a tablet or two different tablets.

[073] For use, the user simply activates the chemistry, in preferred embodiments, by breaking the ampoule (120) and dissolving and mixing the components, then shaking the device or optionally pressing a valve that wets the first carrier material (100). The user then simply removes the cap and wipes the top piece of the device on the contaminated surface. Alternatively, the first carrier material (100) (with the cap open) can be wiped on the wet or moistened surface, on the cap to be closed, on the ampoule to be broken, on the released components to be mixed and distributed by shaking, followed by observation of the color change.

[074] The user can then replace the coating and monitor the color of the first carrier material (100) for up to 5, 10, 15, etc. minutes to detect any color change. Ideally, the entire system is self-contained, with no residue or leakage and presents virtually no hazard to the user. Furthermore, because the first carrier material (100) is made of an adsorbent material, it can effectively collect OPs from the surface with Petition 870210102169, dated 05 / 11 / 2021, pages 291 / 341 29 / 68 very high efficiency. The device takes advantage of the high selectivity of the enzymatic system for the OP / C inhibitor while ignoring almost all environmental interferents and also provides an almost thousandfold signal amplification thanks to the unique dynamic buffering equilibrium response mechanism.

[075] In preferred embodiments, the device is 68 mm high X 50 mm wide. In the final optimized pen, some of the enzymes, substrates, ampoule and dye may be in different compartments but the chemistry may be the same.

[076] To implement and activate the chemistry, the user simply breaks the ampoule (120) by holding the intermediate piece (130) with one hand and twisting the bottom piece (140) 90 degrees with the other hand; dissolving the chemicals contained in the second carrier material, for example, dry paper (urea), a pH-sensitive yellow to red colorimetric dye and enzymatic substrates, for example, 4-nitrophenol acetate (4-NPA). After breaking the ampoule (120), the device is inverted and gravity and some gentle agitation mix the buffer with the second carrier material containing the substrates and then the first carrier material (100) with the incorporated OP / C detection enzyme while the cap is still in place. The user then opens the cap, rubs the contaminated surface with the inverted pen; pressing the first carrier material (100) several times to moisten the first carrier material (100) and ensure proper sampling.The cover is replaced and the color of the first carrier material is monitored for 5 to 10 minutes to detect any change. The entire system is self-contained, with no residue or leakage, and presents virtually no danger to the user. Furthermore, the first carrier material is made of adsorbent material that absorbs and removes the chemicals. Petition 870210102169, dated 05 / 11 / 2021, pages 292 / 341 30 / 68 of the surface with very high efficiency. The device takes advantage of the high selectivity of the enzymatic system for the OP / C inhibitor while ignoring almost all environmental interferents and also provides an amplification of almost a thousand times the signal thanks to the unique dynamic buffering equilibrium response mechanism. C. Reaction used to detect OP / C

[077] In the absence of OP / Cs, the device relies on an enzymatic reaction catalyzed by an OP / C detection enzyme on a first substrate that results in acidic reaction products to lower the pH. Simultaneously, the second reaction system comprising the second enzyme and a second substrate produces basic reaction products that increase the pH. The reaction rates of the first and second reactions are adjusted such that the net change in pH is zero or decreases slightly. This adjusts the reference pH. However, if OPs are present on a surface and are transferred to the first carrier material of the device, the OP / C detection enzyme is inhibited and unable to convert the first substrate and thus lower the pH. Thus, inhibition of the OP / C detection enzyme by a pesticide or OP / C results in a net increase in the system pH above the reference pH.Because a pH-dependent dye is present in the device, a change in pH can be reported by a change in color.

[078] For example, US 6,861,252 (incorporated in the present invention by reference in its entirety) teaches that a pH-responsive colorimetric dye rapidly changes from yellow to red when OP / C nerve agents are detected (see, for example, Figure 2 of this patent). When the OP / C detection enzyme (this patent exemplifies AChE) is inhibited by an OP, substrate hydrolysis and the simultaneous decrease in pH are interrupted, and the second base-producing enzyme continues to form ammonia leading Petition 870210102169, dated 05 / 11 / 2021, pages 293 / 341 31 / 68 the pH of the system dramatically, for example, from 5 to 8. A pH-responsive colorimetric dye (pKa 6.5) is then titrated from yellow to red, providing a localized visual assessment of the presence of OP / C. As described in the present invention, this colorimetric reaction can be used in combination with the device to detect OP / C.

[079] As a further example, nitrazine yellow dye can be used in the device described in the present invention to detect OPs. For example, a nitrazine yellow dye can be incorporated into the first carrier material composing the first carrier material. In preferred embodiments, the first synthesized carrier material has a dye content of approximately 0.4 mg of dye / g of dry polymer. Differences in physical property clearly visible to the naked human eye occur when the polymers were incubated within aqueous solutions of varying pH. The color of the samples varies from bright orange at pH 6.5 to blue at pH 9.0. Distinctions in color were clearly discernible to the naked eye between each of the samples exposed to a pH of 6, 6.4, 6.8, 7.2, 7.6, 8, 8.5 and 9.The color series observed in the polymers of the present invention was the same as the color series that is produced by suspending the soluble dye within the aqueous solution (for example, in the ampoule (120)) over the same pH range.

[080] Furthermore, an approach proposed in the present invention is the use of CES instead of AChE or BChE as the OP / C detection enzyme. We have found that the inhibition constants for OP / C insecticides are much higher (100 to 1000 times) for CES than for AChE. Thus, CES enzymes are preferably used in the device to detect OP / C insecticides.

[081] Additionally, variants can be created using standard mutational tools to generate improved variants that have enhanced sensitivity to different forms of OP / C insecticides in order to be inhibited Petition 870210102169, dated 05 / 11 / 2021, pages 294 / 341 32 / 68 at lower concentrations of OP / C pesticides compared to the protein from which the variant is derived. For example, the proposed enzymes and mutants that can be used to detect OP / C pesticides are selected from: a. Wild-type carboxylesterase aE7 from the Australian blowfly Lucilia cuprina (LcaE7); b. mutant form of LcaE7G137D c. LcaE7 mutants E183, K275, E78 and / or E292 d. Wild-type AChE; e. AChE mutant, such as rHuAChE containing two mutations in the acyl pocket residues (F295L, F297V); f. Carboxylesterase (Cqestβ2) from the mosquito Culex quinquefasciatus g. Any of the enzymes listed in Tables 2 to 5.

[082] Specifically, one approach is to produce wild-type blowfly LcoE7 CES and mutated forms of LcoE7 (e.g., LcαE7G137D). See GenBank Access Q25252_LUCCU for the wild-type sequence. It should be noted that recombinant LcoE7 produced in the E. coli system is monomeric and dimeric while native human CES is trimeric. To examine how trimerization occurs, crystal structures of trimeric human CES produced in hCES1 derived from HEK293 were studied by de Sousa et al. who revealed that trimers were generated by space group symmetry with the K78:E183 and K275:E292 salt bridges. Since the LcoE7 sequence contains E183 and K275 but not E78 or E292, an Lc«E7 mutant expressing all four of these amino acids was produced in order to generate trimers with potentially increased stability. Petition 870210102169, dated 05 / 11 / 2021, pp. 295 / 341 33 / 68 Table 2 - Enzymes Classified as EC 3.1.1.8 Entry Entry Name Protein Names Organism Q95000 CHLE1_BRALA Cholinesterase 1 (Fragment) Branchiostoma lanceolatum (Common lancelet) (Amphioxus lanceolatum) Q95001 CHLE2_BRALA Cholinesterase 2 (Fragment) Branchiostoma lanceolatum (Common lancelet) (Amphioxus lanceolatum) P06276 CHLE_HUMAN Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Pseudocholinesterase) Homo sapiens (Human) P32751 CHLE_MACMU Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Pseudocholinesterase) (Fragment) Macaca mulatto (Rhesus monkey) Q5UR02 CHLE_MIMIV Probable cholinesterase (Acylcholine acylhydrolase) Acanthamoeba polyphaga mimivirus (APMV) P32749 CHLE_BOVIN Cholinesterase (Acylcholine acylhydrolase) (Butyrcholine esterase) (Choline esterase II) (Pseudocholinesterase) Bos taurus (Bovine) P32750 CHLE_CANLF Cholinesterase (Acylcholine acylhydrolase) (Butyrcholine esterase) (Cholineesterase II) (Pseudocholinesterase) (Fragment) Canis lupus familiaris (Dog) (Canis familiaris) O62760 CHLE_FELCA Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Pseudocholinesterase) Felis catus (Cat) (Felis silvestris catus) P81908 CHLE_HORSE Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (EQ-BCHE) (Pseudocholinesterase) Equus caballus (Horse) Q03311 CHLE_MOUSE Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Pseudocholinesterase) Mus musculus (Mouse) O62761 CHLE_PANTT Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Pseudocholinesterase) Panthera tigris (Bengal tiger) Petition 870210102169, dated 05 / 11 / 2021, pages 296 / 341 34 / 68 Table 2 - Enzymes Classified as EC 3.1.1.8 Entry Entry Name Protein Names Organism P32752 CHLE_PIG Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Peudocholinesterase) (Fragment) Sus scrofa (Pig) P21927 CHLE_RABIT Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Peudocholinesterase) Oryctolagus cuniculus (Rabbit) P32753 CHLE_SHEEP Cholinesterase (Acylcholine acylhydrolase) (Butyrylcholine esterase) (Choline esterase II) (Peudocholinesterase) (Fragment) Ovis aries (Sheep) Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism A1CFK9 PATB_ASPCL Carboxylesterase patB (Patulin synthesis protein B) patB ACLA_093570 Aspergillus clavatus (strain ATCC 1007 / CBS 513.65 / DSM 816 / NCTC 3887 / NRRL 1) A0A075TX Z3 PATB_PENEN Carboxylesterase patB (Patulin biosynthesis cluster protein B) patB PEX2_082800 Penicillium expansum (Blue mold rot fungus) D4AV38 LIP4_ARTBC Probable secreted lipase ARB_00047 ARB_00047 Arthroderma benhamiae (strain ATCC MYA4681 / CBS 112371) (Trichophyton mentagrophytes) Q4F883 SG101_ARATH Senescence-associated carboxylase 101 SAG101 At5g14930 F2G14.50 Arabidopsis thaliana (Mouse cress) O31452 YBFK_BACSU YbfK ybfK carboxylesterase BSU02260 Bacillus subtilis (strain 168) Q8VCT4 CES1D_MOUSE 1D-carboxylesterase (Carboxylesterase 3) (EC 3.1.1.67) Ces1d Ces1 Ces3 Mus musculus (Mouse) Petition 870210102169, dated 05 / 11 / 2021, pp. 297 / 341 35 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism (Fatty acid ethyl ester synthase) (FAEE synthase) (Triacylglycerol hydrolase) (TGH) P16303 CES1D_RAT Carboxylesterase 1D (Carboxylesterase ES-10) (Carboxylesterase 3) (EC 3.1.1.67) (ES-HVEL) (Fatty acid ethyl ester synthase) (FAEE synthase) (Hepatic carboxylesterase 10) (pI 6,1 esterase) Ces1d Ces3 Rattus norvegicus (Mouse) Q91WU0 CES1F_MOUSE Carboxylesterase 1F (Carboxylic ester hydrolase) (Triacylglycerol hydrolase 2) (TGH2) Ces1f CesML1 Mus musculus (Mouse) Q9SMM9 CXE13_ARATH Probable carboxylesterase 13 (AtCXE13) CXE13 At3g48700 T8P19.210 Arabidopsis thaliana (Mouse cress) Q9LVB8 CXE20_ARATH Probable carboxylesterase 120 (AtCXE20) CXE20 At5g62180 MMI9.26 Arabidopsis thaliana (Mouse cress) Q9SX78 CXE2_ARATH Probable carboxylesterase 2 (AtCXE2) CXE2 At1g47480 F16N3.25 Arabidopsis thaliana (Mouse Cress) Q9FX92 CXE3_ARATH Probable carboxylesterase 3 (AtCXE3) CXE3 At1g49640 F14J22.12 Arabidopsis thaliana (Mouse Cress) Q9FG13 CXE15_ARATH Probable carboxylesterase 15 (AtCXE15) CXE15 At5g06570 F15M7.10 Arabidopsis thaliana (Mouse Cress) Q9FX93 CXE4_ARATH Probable mitochondrial carboxylesterase 4 CXE4 At1g49650 F14J22.21 Arabidopsis thaliana (Mouse Cress). Petition 870210102169, dated 05 / 11 / 2021, pages 298 / 341 36 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism (AtCXE4) Q9LK21 CXE11_ARATH Probable carboxylesterase 11 (AtCXE11) CXE11 At3g27320 K17E12.14 Arabidopsis thaliana (Mouse cress) Q9SMN0 CXE12_ARATH Probable carboxylesterase 12 (AtCXE12) CXE12 At3g48690 T8P19.200 Arabidopsis thaliana (Mouse cress) Q8LED9 CXE16_ARATH Probable carboxylesterase 16 (AtCXE16) CXE16 At5g14310 F18O22.100 Arabidopsis thaliana (Rat cress) Q9LFR7 CXE17_ARATH Probable carboxylesterase 17 (AtCXE17) CXE17 At5g16080 F1N13.220 Arabidopsis thaliana (Rat cress) Q9LT10 CXE18_ARATH Probable carboxylesterase 18 (AtCXE18) CXE18 At5g23530 MQM1.21 Arabidopsis thaliana (Rat cress) O64641 CXE9_ARATH Probable carboxylesterase 9 (AtCXE9) CXE9 At2g45610 F17K2.14 Arabidopsis thaliana (Rat cress) Q0ZPV7 CXE1_ACTER Carboxylesterase 1 (AeCXE1) CXE1 Actinidia eriantha (Velvet vine) (Actinidia fulvicoma var.lanata) Q9LMA7 CXE1_ARATH Probable carboxylesterase 1 (AtCXE1) CXE1 At1g19190 T29M8.6 Arabidopsis thaliana (Mouse cress) Q9FX94 CXE5_ARATH Probable carboxylesterase 5 (AtCXE5) CXE5 At1g49660 F14J22.11 Arabidopsis thaliana (Mouse cress) Q9SX25 CXE6_ARATH Probable carboxylesterase 6 (AtCXE6) CXE6 At1g68620 F24J5.14 Arabidopsis thaliana (Mouse cress) Q9ZQ91 CXE7_ARATH Probable carboxylesterase 7 (AtCXE7) CXE7 At2g03550 T4M8.1 Arabidopsis thaliana (Mouse cress) O64640 CXE8_ARATH Probable carboxylesterase 8 (AtCXE8) CXE8 At2g45600 17K2.13 Arabidopsis thaliana (Mouse cress) B3PI89 BIOHC_CELJU Bifunctional protein of bioC biosynthesis CJA_0428 Cellvibrio japonicus (Ueda107 strain). Petition 870210102169, dated 05 / 11 / 2021, pages 299 / 341 37 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism Biotin BioHC [Includes: Carboxylesterase BioH (Biotin synthesis protein BioH); Malonyl[acyl-carrier protein] Omethyltransferase (Malonyl-ACP Omethyltransferase) (EC 2.1.1.197) (Biotin synthesis protein BioC)] (Pseudomonas fluorescens subsp. cellulosa) Q21FY5 BIOHC_SACD2 Bifunctional biotin biosynthesis protein BioHC [Includes: Carboxylesterase BioH (Biotin synthesis protein BioH); Malonyl[acyl-carrier protein] O-methyltransferase (Malonyl-ACP O-methyltransferase) (EC 2.1.1.197) (Biotin synthesis protein BioC)] bioC Sde_3137 Saccharophagus degradans (strain 2-40 / ATCC 43961 / DSM 17024) C5BMZ8 BIOHC_TERTT Bifunctional biotin biosynthesis protein BioHC [Includes: Carboxylesterase BioH (Biotin synthesis protein BioH); Malonyl[acyl-carrier protein] O- bioC TERTU_0492 Teredinibacter turnerae (strain ATCC 39867 / T7901) Petition 870210102169, dated 05 / 11 / 2021, pp. 300 / 341 38 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism methyltransferase (Malonyl-ACP O-methyltransferase) (EC 2.1.1.197) (Biotin synthesis protein BioC)] Q5NUF3 HIDH_SOYBN 2-hydroxyisoflavanone α-dehydratase (EC 4.2.1.105) (HIDH Carboxylesterase) HIDH Glyma01g45020 Glycine max (Soybean) (Glycine hispida) Q5NUF4 HIDM_GLYEC 2-hydroxyisoflavanone α-dehydratase (EC 4.2.1.105) (HIDM Carboxylesterase) HIDM Glycyrrhiza echinata (Licorice) P81098 SFAH_HELAN Seed fatty acyl ester hydrolase (Fragment) Helianthus annuus (Common sunflower) P23141 EST1_HUMAN Hepatic carboxylesterase 1 (Acylcoenzyme A:cholesterol acyltransferase) (ACAT) (Cerebral carboxylesterase hBr1) (Carboxylesterase 1) (CE-1) (hCE-1) (Cocaine carboxylesterase) (Egasyn) (HMSE) (Methylumbelliferyl acetate deacetylase 1) (EC 3.1.1.56) (Monocyte / macrophage serine esterase) (Retinyl ester CES1 CES2 SES1 Homo sapiens (Human) Petition 870210102169, dated 05 / 11 / 2021, pp. 301 / 341 39 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism hydrolase) (REH) (Serine esterase 1) (Triacylglycerol hydrolase) (TGH) O46421 EST1_MACFA Hepatic carboxylesterase 1 CES1 Macaca fascicularis (Crab-eating macaque) (Cynomolgus macaque) Q64419 EST1_MESAU Hepatic carboxylesterase Mesocricetus auratus (Syrian hamster) Q5RCL7 EST3_PONAB Carboxylesterase 3 (Hepatic carboxylesterase homolog 31) CES3 Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) P81429 EST1_SCHGA Esterase SG1 (Carboxylic ester hydrolase) (Fragment) SG1 Schizaphis graminum (Green stink bug) Q47M62 EST1_THEFY Carboxylesterase Tfu_2427 Thermobifida fusca (YX strain) Q64573 EST4_RAT Hepatic carboxylesterase 4 (Carboxylesterase ES-4) (Renal microsomal carboxylase) (Microsomal palmitoylCoA hydrolase) Rattus norvegicus (Rat) P25727 EST5A_DROPS Esterase-5A (Est5A) (Carboxylic ester hydrolase 5A) (Carboxylesterase 5A) Est-5A Est5A GA23705Drosophila pseudoobscura pseudoobscura (Fruit fly) Q8I034 EST5A_FELCA Carboxylesterase 5A (Urinary protein excreted similar to carboxylesterase) (Cauxin) CES5A CES7 Felis catus (Cat) (Felis silvestris catus) O00748 EST2_HUMAN Cocaine esterase CES2ICE Homo sapiens Petition 870210102169, dated 05 / 11 / 2021, pages 302 / 341 40 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism (EC 3.1.1.84) (Carboxylesterase 2) (CE-2) (hCE-2) (Methylumbelliferyl acetate deacetylase 2) (EC 3.1.1.56) (Human) Q3T930 EST5A_SHEEP Carboxylesterase 5A (Carboxylesterase-like urinary excreted protein homolog) (Cauxin) (Fragment) CES5A CES7 Ovis aries (Sheep) O16170 EST5B_DROMI Esterase-5B (Est5B) (Carboxylic ester hydrolase 5B) (Carboxylesterase 5B) Est-5B Est5B Drosophila miranda (Fruit flies) Q64176 EST1E_MOUSE Carboxylesterase 1E (Egasyn) (Hepatic carboxylesterase 22) (Es22)(Esterase-22) Ces1e Es22 Mus musculus (Mouse) Q63108 EST1E_RAT Carboxylesterase 1E (Carboxylesterase ES-3) (ES-HTEL) (Egasyn) (Hepatic carboxylesterase 3) (pI 5,5 esterase) Ces1e Ces1 Rattus norvegicus (Rat) P16854 EST1_CULPI Esterase B1 B1 Culex pipiens (House mosquito) Q6UWW8 EST3_HUMAN Carboxylesterase 3 (Hepatic carboxylesterase 31 homolog) CES3 UNQ869 / PRO1887 Homo sapiens (Human) Q51758 EST1_PSEFL Carboxylesterase 1 (Esterase I) estA Pseudomonas fluorescens P10094 EST4_DROMO Esterase-4 Est-4 Est4 Drosophila. Petition 870210102169, dated 05 / 11 / 2021, pages 303 / 341 41 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism (Fragment) mojavensis (Fruit fly) O16173 EST5A_DROPE Esterase-5A (Est5A) (Carboxylic ester hydrolase 5A) (Carboxylesterase 5A) Est-5A Est5A Drosophila persimilis (Fruit fly) Q07085 EST2_CAEEL Esterase CM06B1 F13H6.3 Caenorhabditis elegans Q6NT32 EST5A_HUMAN N Carboxylesterase 5A (Urinary excreted carboxylesterase-like protein homolog) (Cauxin) CES5A CES7 Homo sapiens (Human) Q5GRG2 EST5A_RAT Carboxylesterase 5A (Urinary excreted carboxylesterase-like protein homolog) (Cauxin) (Epididymis-specific gene protein 615) Ces5a Ces7 Rattus norvegicus (Mouse) Q53547 EST2_PSEFL Carboxylesterase 2 (Esterase II) estB Pseudomonas fluorescens O16171 EST5C_DROPE Esterase-5C (Est5C) (Carboxylic ester hydrolase 5C) (Carboxylesterase 5C) Est-5C Est5C Drosophila Drosophila persimilis (Fruit fly) P47982 EST6_DROMA Esterase 6 (Est-6) (Carboxylic ester hydrolase 6) (Carboxylesterase 6) Est-6 est6 Drosophila mauritiana (Fruit fly) Q08662 EST6_DROSI Esterase 6 (Est-6) (Carboxylic ester hydrolase 6) (Carboxylesterase- Est-6 est6 Drosophila simulans (Fruit fly). Petition 870210102169, dated 05 / 11 / 2021, pages 304 / 341 42 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism 6) O33407 ESTA_PSEAE Esterase EstA (EstA autotransporter esterase) estA papA PA5112 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) P81012 ESTA_SCHGA 52 kDa esterase subunit (Carboxylic ester hydrolase) (Fragment) Schizaphis graminum (Green stink bug) P81011 ESTB_SCHGA 56 kDa esterase subunit (Carboxylic ester hydrolase) (Fragment) Schizaphis graminum (Green stink bug) Q9WYH1 ESTD_THEMA Esterase EstD estD TM_0336 THEMA_03040 Tmari_0334 Thermotoga maritima (strain ATCC 43589 / MSB8 / DS M 3109 / JCM 10099) A4KX74 ESTE_HVAVE Putative esterase ORF19 Heliothis virescens ascovirus 3e (HvAV-3e) P35501 ESTE_MYZPE Esterase E4(Carboxylic ester hydrolase) Myzus persicae (Green peach aphid) (Aphis persicae) Q0E588 ESTE_SFAVA Putative esterase ORF13 Spodoptera frugiperda ascovirus 1a (SfAV-1a) P18167ESTP_DROME Esterase P (Est-P) (Carboxylic ester hydrolase P) (Carboxylesterase- Est-P EstP CG17148 Drosophila melanogaster (Fruit fly) Petition 870210102169, dated 05 / 11 / 2021, pages 305 / 341 43 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism P) Q06174 EST_GEOSE Carboxylesterase est est30 Geobacillus stearothermophil us (Bacillus stearothermophil us) P23953 EST1C_MOUSE Carboxylesterase 1C (Hepatic Carboxylesterase N) (Pulmonary Surfactant Convertase) (PESN) Ces1c Es1 Mus musculus (Mouse) P10959 EST1C_RAT Carboxylesterase 1C (Carboxylesterase ES-1) (E1) (ESTHET) (Esterase-2) (Hepatic Carboxylesterase 1) (Neutral Retinyl Ester Hydrolase) (NREH) (Retinyl Ester Hydrolase) (REH) Ces1c Es2 Rattus norvegicus (Rat) Q04456 EST1_CAEBR Intestinal esterase 1 (Non-specific carboxylesterase) ges-1 CBG06418 Caenorhabditis briggsae Q04457 EST1_CAEEL Intestinal esterase 1 (Non-specific carboxylesterase) ges-1 R12A1.4 Caenorhabditis elegans Q63880 EST3A_MOUSE Carboxylesterase 3A (ES-male) (Hepatic carboxylesterase 31) (Esterase-31) Ces3a Es31 Mus musculus (Mouse) Q8VCU1 EST3B_MOUSE Carboxylesterase 3B (Similar to hepatic carboxylesterase 31) Ces3b Gm4738 Mus musculus (Mouse) Q8VCC2 EST1_MOUSE Hepatic carboxylesterase 1 (Acyl- Ces1 Ces1g Mus musculus (Mouse). Petition 870210102169, dated 05 / 11 / 2021, pages 306 / 341 44 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism coenzyme A:cholesterol acyltransferase) (Carboxylesterase 1G) (ES-x) Q29550 EST1_PIG Hepatic carboxylesterase (Proline beta-naphthylamidase) (Retinyl ester hydrolase) (REH) Sus scrofa (Pig) P12337 EST1_RABIT Hepatic carboxylesterase 1 (Acyl coenzyme A:cholesterol acyltransferase) Oryctolagus cuniculus (Rabbit) P86325 EST1_THEFU Carboxylesterase Thermobifida fusca (Thermomonospora fusca) Q6AW47 EST5A_CANLF Carboxylesterase 5A (Urinary excreted protein homologue similar to carboxylesterase) (Cauxin) CES5A CES7 Canis lupus familiaris (Dog) (Canis familiaris) O16168 EST5A_DROMI Esterase-5A (Est5A) (Carboxylic ester hydrolase 5A) (Carboxylesterase 5A) Est-5A Est5A Drosophila miranda (Fruit flies) P21370 EST2_CULPI Esterase B2 (Fragment) Culex pipiens (House mosquito) Q6AW46 EST5A_MOUSE Carboxylesterase 5A (Homolog of excreted urinary protein)similar to carboxylesterase) (Cauxin) Ces5a Ces7 Mus musculus (Mouse) P14943 EST2_RABIT Carboxylesterase CES2ICE Oryctolagus Petition 870210102169, dated 05 / 11 / 2021, pages 307 / 341 45 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism Hepatic 2 cuniculus (Rabbit) O16172 EST5B_DROPE Esterase-5B (Est5B) (Carboxylic ester hydrolase 5B) (Carboxylesterase 5B) Est-5B Est5B Drosophila persimilis (Fruit fly) P25726 EST5B_DROPS Esterase-5B (Est5B) (Carboxylic ester hydrolase 5B) (Carboxylesterase 5B) Est-5B Est5B GA14349 Drosophila pseudoobscura (Fruit fly) O16169 EST5C_DROMI Esterase-5C (Est5C) (Carboxylic ester hydrolase 5C) (Carboxylesterase 5C) Est-5C Est5C Drosophila miranda (Fruit flies) P25725 EST5C_DROPS Esterase-5C (Est5C) (Carboxylic ester hydrolase 5C) (Carboxylesterase 5C) Est-5C Est5C GA19955 Drosophila pseudoobscura (Fruit fly) P10095 EST5_DROMO Esterase-5 (Fragment) Est-5 Est5 Drosophila mojavensis (Fruit fly) Q63010 EST5_RAT Hepatic carboxylesterase B-1 (Microsomal hepatic carboxylesterase) Rattus norvegicus (Rat) B2D0J5 EST6_APIME Venom carboxylesterase-6(Api allergen 8) Apis mellifera (Honey bee) P08171 EST6_DROME Esterase-6 (Est-6) (Carboxylic ester hydrolase 6) (Carboxylesterase 6) Est-6 EST6 CG6917 Drosophila melanogaster (Fruit fly) Petition 870210102169, dated 05 / 11 / 2021, pp. 308 / 341 46 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism Q6B6R8 ESTA_PSEPU Esterase EstA estA Pseudomonas putida (Arthrobacter siderocapsulatus) P35502 ESTF_MYZPE Esterase FE4 (Carboxylic ester hydrolase) Myzus persicae (Peach aphid) (Aphis persicae) Q88QS0 ESTP_PSEPK Esterase EstP (EstP autotransporter esterase) (PalmitoylCoA hydrolase) (EC 3,1,2,2) estP PP_0418 Pseudomonas putida (cepa ATCC 47054 / DSM 6125 / NCIMB 11950 / KT2440) Q05487 ESTS_DROVI Esterase S (Est-S) (Éster carboxílico hidrolase S) (Carboxilesterase- S) EstS Drosophila virilis (Mosca-dasfrutas) O32232 EST_BACSU Carboxylesterase est yvaK BSU33620 Bacillus subtilis (onion 168) Q9HZY8 EST_PSEAE Esterase TesA tesA PA2856 Pseudomonas aeruginosa (onion ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) P9WK87 NLHH_MYCTU Carboxylesterase NlhH nlhH lipH Rv1399c Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) O06350 LIPF_MYCTU Carboxylesterase LipF lipF Rv3487c Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) L0TC47 LIPV_MYCTU Lipase LipV lipV Rv3203 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv), Petition 870210102169, dated 05 / 11 / 2021, p. 309 / 341 47 / 68 Table 3 - Enzymes Classified as EC 3.1.1.1 Entry Entry Name Protein Names Gene Names Organism P9WK86 NLHH_MYCTO Carboxylesterase NlhH nlhH lipH MT1443 Mycobacterium tuberculosis (CDC strain 1551 / Oshkosh) P96688 NAP_BACSU Uncharacterized carboxylesterase nap nap BSU05440 Bacillus subtilis (strain 168) Table 4 - Enzymes Classified as EC 3.1.1.7 Entry Entry Name Protein Names Gene Names Organism Q867X3 ACES_CULPP Acetylcholinesterase ACE-1 Culex piliens (Northern house mosquito) P04058 ACES_TETCF Acetylcholinesterase Ace Tetronarce californica (Pacific electric ray) (Torpedo californica) Q867X2 ACES_CULQU Acetylcholinesterase ACE-1 Culex quinquefasciatus (Southern house mosquito) (Culex pungens) P38433 ACE1_CAEEL Acetylcholinesterase 1 ace-1 W09B12,1 Caenorhabditis elegans P21836 ACES_MOUSE Acetylcholinesterase Ache Mus musculus (Mouse) P56161 ACES_ANOST Acetylcholinesterase Anopheles stephensii (Indo-Pakistani malaria mosquito) P07140 ACES_DROME Acetylcholinesterase Ace CG17907 Drosophila melanogaster (Fruit fly) O62763 ACES_FELCA Acetylcholinesterase ACHE Felis catus (Cat) (Felis silvestris catus) P36196 ACES_CHICK Acetylcholinesterase ACHE Gallus gallus (Chicken) Q92081 ACES_MYXGL Acetylcholinesterase ache ace1 Myxine glutinosa (Atlantic hagfish) Q869C3 ACES_ANOGA Acetylcholinesterase Ace ACE1, ACHE1, AGAP001356 Anopheles gambiae (African malaria mosquito) Q27459 ACE1_CAEBR Acetylcholinesterase 1 ace-1 CBG16374 Caenorhabditis briggsae O42275 ACES_ELEEL Acetylcholinesterase Ache Electrophorus electricus (Electric eel), Petition 870210102169, dated 05 / 11 / 2021, pages 310 / 341 48 / 68 Table 4 - Enzymes Classified as EC 3.1.1.7 Entry Entry Name Protein Names Gene Names Organism (Gymnotus electricus) P07692 ACES_TORMA Acetylcholinesterase Ache Torpedo marmorata (Marbled electric ray) P23795 ACES_BOVIN Acetylcholinesterase ACHE Bos taurus (Cattle) Q86GC8 ACES_CULPI Acetylcholinesterase ACHE1 Culexpipiens (House mosquito) Q27677 ACES_LEPDE Acetylcholinesterase Leptinotarsa ​​decemlineata (Colorado potato beetle) (Doryphora decemlineata) P37136 ACES_RAT Acetylcholinesterase Ache Rattus norvegicus (Mouse) Q86GC9 ACES_CULTO Acetylcholinesterase ACE-1 Culex torrentium (Mosquito) W4VSJ0 ACES_TRILK Acetylcholinesterase-1 Trittame loki (Trapdoor spider) Q9DDE3 ACES_DANRE Acetylcholinesterase Ache Danio rerio (Zebrafish) (Brachydanio rerio) P22303 ACES_HUMAN Acetylcholinesterase ACHE Homo sapiens (Human) Q7LZG1 ACES_NAJOX Acetylcholinesterase ACHE Naja oxiana (Central Asian cobra) (Oxus cobra) Q9NDG8 ACE4_CAEBR Acetylcholinesterase 4 ace-4CBG02827 Caenorhabditis briggsae Q29499 ACES_RABIT Acetylcholinesterase ACHE Oryctolagus cuniculus (Rabbit) Q92035 ACES_BUNFA Acetylcholinesterase ACHE Bungarus fasciatus (Banded Krait) (Pseudoboa fasciata) Table 5 - Mutations in Lucilia cuprina Mutation Comments Literature E217M mutant at anionic site, p1 subsite, pyrethroid hydrolysis similar to wild type 668944 F309L mutant at p2 acyl pocket subsite, marked increase in pyrethroid hydrolysis for cis substrate, strong increase for trans substrate 668944 Petition 870210102169, dated 05 / 11 / 2021, pp. 311 / 341 49 / 68 Table 5 - Mutations in Lucilia cuprina Mutation Comments Literature F354L mutant at anionic site, p1 subsite, pyrethroid hydrolysis similar to wild type 668944 F354W mutant at anionic site, p1 subsite, marked increase in pyrethroid hydrolysis for both cis and trans substrates 668944 G137D mutant at oxyanion hole, marked decrease in pyrethroid hydrolysis 668944 G137E mutant at oxyanion hole, strong decrease in pyrethroid hydrolysis 668944 G137H mutant at oxyanion hole, marked decrease in pyrethroid hydrolysis 668944 G137R mutant at oxyanion hole, pyrethroid hydrolysis similar to wild type 668944 M364L / I419 F / A472T / I50 5T / K530E / D554G mutant shows increased activity 730817 W251A mutant in acyl pocket p2 subsite, marked increase in pyrethroid hydrolysis for both cis and trans substrate 668944 W251G mutant in acyl pocket p2 subsite,Marked increase in pyrethroid hydrolysis for both cis and trans substrates. 668944 W251L mutant in acyl pocket p2 subsite, strong increase in pyrethroid hydrolysis for both cis and trans substrates. The trans:cis ratio for substrate preference is 2:1 compared to 27:1 in the wild type. 668944 W251L / D449 G mutant shows a loss of activity for most substrates. 729826 W251L / D473 N mutant shows a loss of activity for most substrates. 729826 W251L / F309 L mutant in acyl pocket p2 subsite, marked increase in pyrethroid hydrolysis for both cis and trans substrates. The trans:cis ratio for substrate preference is 2:1 compared to 27:1 in the wild-type 668944 W251L / G137 D mutant in the acyl pocket p2 subsite.A marked increase in pyrethroid hydrolysis for both cis and trans substrates. The trans:cis ratio for substrate preference is 2:1 compared to 27:1 in the wild type. 668944 W251L / I140 F mutant shows a loss of activity for most substrates. 729826 W251L / I459 N mutant shows a loss of activity for most substrates. 729826 W251L / P250 S mutant in acyl pocket p2 subsite, marked increase in pyrethroid hydrolysis for both cis and 668944 substrates. Petition 870210102169, dated 05 / 11 / 2021, pages 312 / 341 50 / 68 Table 5 - Mutations in Lucilia cuprina Mutation Comments Literature trans. The trans:cis ratio for substrate preference is 3:1 compared to 27:1 in the wild type. W251L / R458 C the mutant shows a loss of activity for most substrates. 729826 W251L / R461 H the mutant shows a loss of activity for most substrates. 729826

[083] As described in the present invention, the conversion of the second substrate by the second enzyme results in the basification of the reaction buffer. Representative examples of a second substrate and second enzyme include, but are not limited to, urea and urease (classified as EC 3.5.1.5), urea and urea amidolyase (classified as EC 6.3.4.6 and EC 3.5.1.54), biuret and biuret amidohydrolase (classified as EC 3.5.1.84), [beta-hydroxypyruvate + glycolaldehyde] and transketolase (classified as EC 2.2.1.1, with representative examples of substrates being: D-fructose 6-phosphate, D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate or D-xylulose 5-phosphate), adenosine and adenosine deaminase (classified as EC 3.5.4.4), adenine and adenine deaminase (classified as EC 3.5.4.15), guanosine and guanosine deaminase (classified as EC 3.5.4.15), guanine and guanine deaminase (classified as EC 3.5.4.3), cytosine and cytosine deaminase (classified as EC 3.5.4.5).

[084] In addition, representative second enzyme / second substrate combinations can be selected from those shown in Table 6.

[085] In preferred embodiments, ureases are used as the second enzyme. Ureases (EC 3.5.1.5) are highly homologous nickel-dependent enzymes predominantly found in plants, bacteria, and fungi, which hydrolyze urea into ammonia and carbon dioxide [1, 2]. Plant and fungal ureases are homotrimers or hexamers of a ~90 kD subunit, while ureases Petition 870210102169, dated 05 / 11 / 2021, pages 313 / 341 51 / 68 bacterial ureases are multimers of two- or three-subunit complexes [3-4]. The N-terminal halves of single-chain plant or fungal urease align with the primary sequence of the small subunits of most bacterial enzymes (e.g., β and γ chains of Bacillus pasteurii urease or the A subunit of Helicobacter pylori urease). The C-terminal portions of plant and fungal chains resemble the large subunits of bacterial ureases (e.g., the α chain of B. pasteurii urease or the B subunit of H. pylori enzyme). Considering the similarity in their sequences, all ureases likely possess similar tertiary structures and catalytic mechanisms, indicating that they are variants of the same ancestral protein [2]. H. pylori urease (1E9Z) and jack bean (Canavalia ensiformis) major urease (P07374) share approximately 50% identity despite differences in their quaternary structures.The 3D crystallographic structures of three bacterial ureases were successfully solved: Klebsiella aerogenes (1FWJ), B. pasteurii (4UBP) and H. pylori (1E9Z). Petition 870210102169, dated 05 / 11 / 2021, pages 314 / 341 52 / 68 Table 6 Enzyme Substrate Reaction Products Enzyme Class Reference(s) Examples of Seq-ID urease urea CO2 + 2 NH3 3.5.1.5 Balasubramanian 2010; Wassermann 2010; Kappaun 2018; Filiz 2016 P07374, I1K3K3, 1FWJ, 4UBP, 1E9Z Allophanate hydrolase Allophanate (1) CO2 + 2 NH3 3.5.1.54 Zhao 2018 Q936X2, 4CP8 Urea amidolyase Urea + ATP + HCO3- 2 CO2 + 2 NH3 6.3.4.6 + 3.5.1.54 Zhao 2018 Biuret amidohydrolase biuret urea + CO2 + NH3 3.5.1.84 Esquirol 2018 A0A075T5U3, Q1M7F4 Transketolase β-hydroxypyruvate + glycolaldehyde L-erythrulose + CO2 2.2.1.1 Gruber 2017 Adenosine deaminase Adenosine Inosine + NH3 3.5.4.4 Alberty 2007 P00813, P22333, Adenine deaminase Adenine Hypoxanthine + NH3 3.5.4.2 Guanosine deaminase Guanosine Xanthosine + NH3 3.5.4.15 P76641 Guanine deaminase (Cypin) Guanine Bitra 2013a, Bitra 2013b Q82Y41 Cytidine deaminase Cytidine Uridine + NH3 3.5.4.5 Dong 2015 P0ABF6 Petition 870210102169, dated 05 / 11 / 2021, pages 315 / 341 53 / 68

[086] Changes in pH can be measured using standard techniques known in the art. For example, techniques such as those described in Gruber et al., “Real-time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side-entry reactor (μSER) shows potential for pH control” Biotechnology Journal, Vol. 12:6 (June 2017) can be used. In this example, enzymatic activity was determined by mixing 250 pL of a 100 mM lithium-hydroxypyruvate (HPA) and 100 mM glycolaldehyde (GA) solution with 250 pL of a transketolase lysate solution (250 pL TK lysate, 4.8 mM thiamine diphosphate ThDP, and 19.6 mM magnesium chloride MgCb). Both solutions were prepared in 50 mM Tris-HCl buffer at pH 7.0.

[087] In another example, biuret hydrolase can also be used to measure pH change as described in Esquirol et al. Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841 PLOS / ONE (2018). In the present invention, the specific biuret hydrolase activity was obtained using 22 nM of wild-type biuret hydrolase or 0.22 μM of variants and 5 mU / pL of GDH in the presence of 1.2 mM biuret in 25 mM potassium phosphate buffer at pH 8.5, at 28 °C. Kinetic data for biuret were measured for the wild type and all variants having a residual specific activity above 1% of the wild-type enzymes, using 22 nM biuret hydrolase enzyme and 2.9 pM or 0.9 pM of the variants, depending on their performance in the presence of various biuret concentrations ranging from 0 to 4 mM, using the GDH-coupled assay.All kinetic constants were calculated using GraphPad Prism (GraphPad Software, San Diego, USA) by fitting the rate data to the Michaelis-Menten equation. Petition 870210102169, dated 05 / 11 / 2021, pages 316 / 341 54 / 68 D. Device used to detect OP phosphorothionate tion forms

[088] However, unlike OP nerve agents, which are potent inhibitors in their native non-activated forms, certain phosphorothionate insecticides such as chlorpyrifos, malathion, and parathion must first be converted to replace the P=S bond with a P=O bond, for example, by P450, to generate the active oxon form (e.g., chlorpyrifos oxon (CPO), malathion oxon (MX), and paraoxon (PX)) for their insecticidal action. Furthermore, the ki values ​​obtained for AChE by oxons (e.g., CPO, MO, PX) are 10 to 100 times lower than those of nerve agents (~1.0 x 107 M-^min-1), while the unmodified forms (e.g., malathion) are up to 1,000 times lower.This translates into a very slow color change from yellow to pink (YP) and requires modification of the OP / C detection enzyme (quantity and activity) in the first carrier material of the device to convert the tion to the oxon form in order to increase the reaction rate and produce an efficient device as described in the present invention.

[089] Thus, in order to detect certain OP / C insecticides, the device must also have the ability to convert an OP tion form to the oxon form.

[090] Another approach to obtaining satisfactorily low inhibition constants (e.g., a Ki in the range of 10⁵ to 10⁶ M⁻¹ min⁻¹) includes producing and incorporating P450 (such as, for example, (CYP1A2, CYP6G1)) along with cytochrome c reductase (NAPDH) into the first carrier material, bulb, or second carrier material to enzymatically convert the OP / C pesticide ion to oxon forms. For example, OPs with sufficient inhibition of the OP / C detection enzyme (e.g., Ki = 10⁵ M⁻¹ min⁻¹) can be immediately used in the device. However, OP / C having low inhibition (e.g., Ki = 10¹-3 M⁻¹ min⁻¹), such as in the case of ion forms Petition 870210102169, dated 05 / 11 / 2021, pages 317 / 341 55 / 68 of OP / C insecticides) will need conversion to oxon forms chemically (e.g., by chemical oxidants such as halogens (e.g., fluorine, chlorine, bromine, and iodine) or by P450 (plus NADPH). Figure 8 shows the structure of the most commonly used OP / C insecticides in Asia, Central America, India, and the USA, and how (kialta), already containing the P=O linkage and thus highly toxic, represents some of the most widely used toxic OP / C insecticides in these regions (Table 7). Thus, it is anticipated that food samples, for example, from these regions can be readily tested for the presence of OP / C pesticides, at which point the device converts the thion form to the oxon forms. Table 7. Some of the most commonly used OP insecticides in each country. United States Mexico China India Thailand Chlorpyrifos Acephate* Malathion Naled Phorate tophosophos Dicrotophos* Phosmet Dimethoate Terbufos Etoprophos Tetrachlorvinphos Chlorpyrifos Omethoate* Dimethoate* Acephate* Triazophos Methylparathion Monocrotophos ofos* Phorate Leite# Dichlorvos* Phorate Chlorpyrifos, chlorfenvinphos Dichlorvos* Metamidophos* Omethoate* Acephate Dimethoate ofos Isocarb ofos Monocrotophos ofos* Thiazophosophos Phosphamidone Methylparathion Phorate Chlorpyrifos Malathion Monocrotophos ofos* Diazinone Omethoate* tophosophos Dicrotophos* Methylparathion Insecticides are listed according to their use (tons) where it is known. Many of these insecticides are used despite being banned in many countries. Many other, less commonly used insecticides are not listed. • Toxic insecticides from kialtacontra CES tested so far. Petition 870210102169, dated 05 / 11 / 2021, pages 318 / 341 56 / 68 # Widely used as an ectoparasiticide in dairy cattle or in crops used for animal feed, in samples of homogenized and pasteurized Mexican milk.

[091] For example, using a P450 enzyme together with the cofactor NAPDH, the efficiency of enzymatic conversion of the substrate by the OP / C detection enzyme is improved, thereby increasing the ability to detect OPs having high ki. Representative P450 proteins that can be used include, but are not limited to, an example of the P450 enzyme being a triple mutant of CYP1A2 (P450 BM-3 (CYP102-A1)). P450 / NADPH can be included in the second carrier material, within the ampoule, or included within the first carrier material. The expression of P450 CYP6G1 in plants has been described, and thus we intend to explore its internal production in plants. Furthermore, several commercial recombinant cytochrome P450 / NADPH reagents, both human (CYP1A2 (Sigma #C8113 manufactured in baculovirus-infected insect cells; #E9288 expressed in Saccharomyces cerevisiae) and insect (CYP6G1 kindly provided by Dr. Colin Jackson, ANU, Australia) are available and will also be tested.Also, cytochrome P450 (CYP1A2) / NADPH microsomes (Fischer Scientific) are available and were used in Figure 1b. Examples

[092] The present invention will now be further illustrated with reference to the following examples. It will be understood that what follows is by way of example only and that modifications in detail may be made while still falling within the scope of the present invention. Example 1. Production of CES extract derived from plant or purified protein

[093] Representative OP / C detection enzymes, carboxylase Petition 870210102169, dated 05 / 11 / 2021, pp. 319 / 341 57 / 68 human CES1 and CES2 were produced in leaf extract as described below. The constructs were modified using methods and strategies described previously. See, Rosenberg, YJ et al. “A Highly Stable Minimally Processed Plant-Derived Recombinant Acetylcholinesterase For Nerve Agent Detection In Adverse Conditions,” Sci. Rep. 5, 13247; doi: 10.1038 / srepl3247 (2015). Carboxylesterase 1 of Homo sapiens GenBank: BC012418.1)( / protein_id=AAH12418.1) Term 1). Salt bridges K78:E183 and K275:E292 (yellow) 2) C87-C116 & C274-C285 disulfide bridges (green) 3) . N79Q, mutation S221A not present (blue) >hCESl (SEQ ID NO: 5) mwlpalvlatlaasaawg|hpssppvvdtvhgkvlgkfvslegfaqpvaiflgipfa KPPLGPLRFTPPQPAEPWSFVKNATSYPPMCTQLPKELQELQLQLLQLLQLLKECL YLNIYTPADLTKKNRLPVMVWIHGGGLMVGAASTYDGLALAAHENVVVVTIQYRLGIWG FFSTGDEHSRGNWGHLDQVAALRWVQDNIASFGGNPGSVTIFGASAGGESVSVLVLSPL AKNLFHRAISESGVALTSVLVKKGDVKPLAEQIAITAGCKTTTSAVMVHCLRQKTEEELLET TLKMKFLSLDLQGDPRESQPLLGTVIDGMLLLKTPEELQAERNFHTVPYMVGINKQEFG WLIPMLMSYPLSEGQLDQKTAMSLLWKSYPLVCIAKELIPEATEKYLGGTDDTVKKKDLFL DLIADVMFGVPSVIVARNHRDAGAPTYEFQYRPSFSSDMKPKTVIGDHGDELFSVFG APFLKEGASEEEIRLSKMVMKFWANFGNPEGLPYKHQQLQQLQYQL AAQKLKDKEVAFWTN LFAKKAVEKPPQTEHI EL >hCES2 (ACCESS U60553) MSAVACGLLLLLVRGQGQDSASPIRTTHTGQVLGSLVHVKGANAGVQTFLGIPFAK PPLGPLRFAPPEPPESWSGVRDGTTHPAMCLQDLTAVESEFLSQFNMTFPSDSMSEDCL YLSIYTPAHSHEGSNLPVMVWIHGGALVFGMASLYDVRLVRLGVLGVGV DKHATGN WGYLDQVAALRWVQQNI AH FGG N PDRVTIFG ESAGGTSVSSLVVSPIS QGLFHGAIMESGVALLPGLIASSADVISTVVANLSACCDQVDSEALVGCLRGKSKEEILAINK Petition 870210102169, dated 05 / 11 / 2021, pages 320 / 341 58 / 68 PFKMIPGVVDGVFLPRHPQELLASADFQPVPSIVGVNNNEFGWLIPKVMRIYDTQKEMD REASQAALQKMLTLLMLPPTFGDLLREEYIGDNGDPQTLQAQFQEMMADSMFVIPALQ VAHFQCSRAPVYFYEFQHQPSWLKNIRPPHMKADHGDELPFVFRSFFGGNYIKFTEEEEQ LSRKMMKYWANFARNGNPNGEGLPHWPLFDQEEQYLQLNLQPAVGRALKAHRLQFW KKALPQKIQELEEPEERHTEL

[094] Additionally, OP / C detection enzyme constructs comprising human AChE and / or BChE have been generated as previously described in US2017 / 0081649, which is incorporated into the present invention by reference in its entirety. Production of either enzyme can be carried out as follows.

[095] One liter of a modified extraction buffer containing 5 mM MgCb, 4 mM DTT, 150 mM sodium metabisulfite, and 10% sucrose in PBS at pH 7.4 was prepared and refrigerated at 4°C before use. Chitosan was prepared (Chitosan, low molecular weight, Sigma Aldrich 448869 - 50 g) by adding 1% w / v chitosan to 1% acetic acid and stirring the solution for at least 30 minutes until dissolved and assuming a gelatinous appearance. Frozen leaves were ground in a Vitamix blender with 5X w / v of extraction buffer. After grinding, the fluid paste was passed through Miracloth (Calbiochem #475855), poured into centrifuge bottles, and centrifuged at 20,000 xg for 15 minutes. After centrifugation, the supernatant was poured into a beaker, the pH was changed to 7.4, and chitosan was added at 0.2% v / v. The extract containing chitosan was then stirred at 4 °C for 30 minutes, removed from the stirrer, and left for an additional 30 minutes at 4 °C.The extract was poured into centrifuge bottles and centrifuged at 1500 rpm in a Sorvall RT6000 chilled to 4°C for 5 minutes. The supernatant was decanted and left at 4°C until the enzyme level was determined. In some cases, collagen hydrolysate was added to the extract beforehand. Petition 870210102169, dated 05 / 11 / 2021, pages 321 / 341 59 / 68 was aliquoted and frozen at -20 °C.

[096] rHuCES1 was expressed and the extract purified essentially as previously described for AChE (Rosenberg 2015). Briefly, the His-terminally labeled rHuCES1 was expressed by N. b benthamiana using the Agrobacterium leaf infiltration method and extracted from the leaves using a blender and 5 mL of extraction buffer per gram of leaf biomass. The homogenate was filtered through miracloth, clarified by centrifugation, and the pH adjusted to 7.4 before adding chitosan to precipitate phenols, fatty compounds, and other impurities. After a second centrifugation step, the pH was adjusted to 8.0, and DEAE Sephadex A-25 was added to remove other contaminants by negative ion-exchange batch chromatography. The supernatant was 0.45 µm filtered, the pH readjusted to 8.0, centrifuged, and loaded onto a Ni2+-NTA resin.The bound proteins were eluted using step gradients of 30 mM and 100 mM imidazole, and the elution fractions were tested for enzymatic activity. Positive fractions were pooled, concentrated by ultrafiltration, dialyzed against 10 mM Tris at pH 8.0, and stored at 4 °C.

[097] The activity of the OP / C detection enzyme can be determined spectrophotometrically at 25 °C according to the Ellman method. See Ellman et al., 1961, which is incorporated into the present invention by reference. For example, to evaluate AChE activity, the assay mixture contains 1 mM acetylthiocholine as the substrate and 1 mM 5,5-dithiobisnitrobenzoic acid (DTNB) in 50 mM sodium phosphate, pH 8.0, at room temperature (RT). In assays using mammalian cells, 20 μM of ethopropazine is used as a specific BChE inhibitor. BChE activity was similarly evaluated using 1 mM butyrylthiocholine (BTC) as an example substrate and 0.5 mM 5,5-dithiobis2-nitrobenzoic acid (DTNB). This was followed by monitoring the Petition 870210102169, dated 05 / 11 / 2021, pages 322 / 341 60 / 68 increase in absorbance of 5-thio-2-nitrobenzoic acid at 412 nm using a molar extinction coefficient of 14,150 M-1cm-1. One unit of enzymatic activity is defined as the amount required to hydrolyze 1 pmol of substrate / min.

[098] The carboxylesterase activity was assessed by 4-nitrophenyl acetate conversion and determination of the released 4-nitrophenyl by absorbance at 405 nm. Buffer was used as a negative control. Kinetic measurements and Vmax determination were performed on a Spectramax plus 384 microplate reader (Molecular Devices) using Softmax Pro. Several alternative substrates are readily available and will be analyzed for increased turnover rates. Previous studies have shown that 4-nitrophenyl-butyrate is the best substrate for HuCES2 among several 4-nitrophenyl esters [31,33].

[099] Alternatively, the OP / C detection enzyme can be readily produced using a transient expression system of N. benthamiana plant which is inexpensive and can produce kilogram quantities of extract in <2 weeks. See, for example, US 10,221,402 which is incorporated herein by reference in its entirety. Specifically, transient plant expression can generate extracts containing sufficient OP / C detection enzyme activity and purification was not required for OP detection purposes in the device. Recombinant enzymes in supernatants (SN) or extracts can be purified using procainamide sepharose chromatography as previously described (De la Hoz et al., 1986).After loading the SN or extract and washing the column, BChE is usually eluted with a 0.1 to 1 M NaCl gradient, but both AChE and BChE can be efficiently eluted using 0.2 M procainamide, 0.2 M acetylcholine, 0.02 M decamethodium, 0.5 M chlorine chloride, or tetramethyl bromide. Petition 870210102169, dated 05 / 11 / 2021, pages 323 / 341 61 / 68 ammonium at 0.5 M.

[0100] In addition to plant expression, a variety of host expression vector systems can also be used to express the OP / C detection enzyme. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the OP / C detection enzyme. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA, or cosmid DNA containing sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing coding sequences;Insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter);the vaccinia virus promoter (7.5K). Preferably, bacterial cells such as Escherichia coli and, more preferably, eukaryotic cells, are used for the expression of the OP / C detection enzyme. For example, mammalian cells such as ovarian cells; Petition 870210102169, dated 05 / 11 / 2021, pp. 324 / 341 62 / 68 of Chinese hamster (CHO), in combination with a vector such as the main early promoter element of human cytomegalovirus intermediate gene is an effective expression system (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (1990)).

[0101] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use. For example, when a large amount of a protein needs to be produced, vectors that target the expression of high levels of OP / C sensing enzyme that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruther et al., EMBO 1. 2:1791 (1983)), in which the coding sequence can be individually ligated into the vector in the frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express exogenous polypeptides such as glutathione 5-transferase (GST) fusion proteins.In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione agarose matrix beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or Factor Xa protease cleavage sites so that the cloned target gene product (e.g., OP / C detection enzyme) can be released from the GST portion.

[0102] In an insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express an OP / C detection enzyme. The virus grows in Spodoptera frugiperda cells. Encoding sequences can be individually cloned into Petition 870210102169, dated 05 / 11 / 2021, pages 325 / 341 63 / 68 non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0103] In host mammalian cells, several viral-based expression systems that can be used express the OP / C detection enzyme. In cases where an adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, for example, the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo.

[0104] Insertion into a non-essential region of the viral genome (e.g., El or E3 region) will result in a recombinant virus that is viable and capable of expressing the OP / C detection enzyme in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355 - 359 (1984)). Example 2. Bi-molecular rate constants (ki) of AChE inhibition of CES and rHuAChE by the thion and oxon forms of OP insecticides.

[0105] Although the ki for rHuAChE for nerve agents is high (~108M^min-1), the inhibition constants (ki) of rHuAChE for selected OP insecticides (dichlorvos, chlorpyrifos, and malathion) (paraoxon control) were found to be 10 to 1,000 times lower than those for OP nerve agents. By comparison, carboxylesterase (CES) exhibits 10 to 1,000 times higher inhibition parameters for both thion and oxon forms of pesticides than rHuAChE. Thus, in preferred embodiments, HuCES can be used in the device as described in the present invention.

[0106] For example, previously published results showed that the Australian blowfly carboxylesterase Lucilia cuprina (LcaE7) has a high affinity (~5 μM) and kinetic parameters (~1.0 x 107M-1^min-1) for Petition 870210102169, dated 05 / 11 / 2021, pages 326 / 341 64 / 68 a form of OP insecticide tion (17) and that the mutant form of aE7 (LcaE7G137D) had an increased turnover rate by two orders of magnitude for paraoxon hydrolysis. Based on these data, we proposed to include these OP / C Detection Enzymes in the device as described in the present invention.

[0107] Specifically, the human carboxylesterase 1 (CES1) gene (GenBank Accession No. AAH12418.1) and the CES2 gene (GenBank Accession No. AAB03611.1) are transiently produced in N. bentiamiana as described above. Two forms, with and without N-terminal His tags, were compared and purified: the former providing better yields in preliminary studies. Plant-derived rHuCE extracts were tested against a battery of OP insecticides. The results showing different levels of plant rHuCE inhibition by different oxon and tion forms of OP insecticides are shown in Figure 1A.

[0108] Although certain OP insecticides had sufficiently high ki against rHuCE to cause a rapid color change in a PESTpen (~105 M⁻¹min⁻¹), values ​​for many others, e.g., parathion, omethoate, malathion, chlorpyrifos, daizonin, etc., were only 101-3 M⁻¹min⁻¹ and needed to undergo oxidation to convert the thion form to an oxon. In a preliminary in vitro study, the addition of an oxidant, such as CYPP450 / NADPH microsomes (Fischer), increased the ki of parathion 10 times, while chlorpyrifos increased it only slightly. See Figure 1B. These initial data demonstrate that thion conversion can be optimized using an oxidant such as P450 / NADPH.

[0109] In fact, when this experiment was repeated, an increase of more than 50 times was observed. Here, 25 and 50 μE of cytochrome P450 (CYP1A2) (Fischer Scientific and Sigma Aldrich) plus NADPH (1 mM) were added to Petition 870210102169, dated 05 / 11 / 2021, pages 327 / 341 65 / 68 58 pg of parathion and 70 pg of chlorpyrifos, incubated for 10, 20, and 40 min and serially diluted before the addition of rHuCES for an additional 10 min. Figure 1D shows 50-fold increases in the ki of parathion and 20-fold increases for chlorpyrifos after 10 min of incubation with P450 with clear positives at 5.8 and 7 pg respectively. No difference was observed when pre-incubation of OP with P450 was extended to 20 and 40 min, and only small differences were observed using 25 vs 50 pL of P450.

[0110] Figure 1C indicates that the bi-molecular rate constants of plant-derived rHuCE extracts against a battery of OP insecticides were similar to the purified internal rHuCE controls produced in E. coli. Figure 1C also shows that the OP insecticides fell into two groups; those with low ki (101 to 103 M-1^min-1) versus those with high ki (105 M-1^min-1). This was shown to be correlated with their structure, where insecticides, for example, malathion, parathion, chlorpyrifos, exhibiting low ki, had P=S linkages and needed desulfurization for their phosphorylation activity, while dichlorvos, fenamifos, and methamidophos already had a P=O linkage and were already active. Example 3: Device capable of detecting OPs

[0111] In preferred embodiments, the enzymatic components, including the recombinant OP / C Detection Enzymes produced in Example 1, will be manufactured together with a first applicator carrier material, such as, for example, a polyurethane foam applicator sponge, while the first substrate and other additives (coloring compounds, surfactants, rheological thickeners and enzymatic substrates) are kept in a reservoir, i.e., the second carrier material and buffers in a second reservoir, i.e., the ampoule.

[0112] For example, in one embodiment, rHuCE will be incorporated into Petition 870210102169, dated 05 / 11 / 2021, pp. 328 / 341 66 / 68 first carrier material (preferably a polyurethane foam) in the device described in the present invention. The bottom piece (130) of the device contains dry chemicals and a glass ampoule (120) filled with aqueous buffer. The user breaks the ampoule to activate the device, then inverts the device and rotates the cylinder to introduce the wet chemical to the enzymatic foam. Once activated, the cover can then be removed and the first carrier material (100) can be used on sample surfaces.

[0113] In more detail, an OP / C detection enzyme, such as CES, can be co-immobilized onto the first carrier material with nitrazine yellow dye. The first carrier material (150 mg discs) can be incubated (2 mL) with various concentrations of dimethyl methylphosphonate (DMMP) for thirty minutes. A concentrated solution (2 mL) of the first substrate (i.e., 50 mM of a 4-nitrophenyl ester) can then be applied to each first carrier material by breaking the ampoule. Since CES catalyzes the hydrolysis of the first substrate, the pH is reduced and the first carrier material undergoes a transition from bluish-brown to orange. However, if the first carrier material comes into contact with an OP / C, the conversion of the first substrate to acetic acid is inhibited and the color change occurs.

[0114] Once a surface is tested, the lid can then be replaced and the colorimetric scheme (yellow to red) indicates whether any OPs are present within two to 20 minutes (Fig. 2d-4). In preferred embodiments, the shelf life of the enzyme for device products should exceed 60 days when incubated at 37 °C.

[0115] In certain embodiments, both a minimally processed OP / C detection enzyme (including, but not limited to, a plant extract) and the purified protein can be used in the first carrier material. Petition 870210102169, dated 05 / 11 / 2021, pp. 329 / 341 67 / 68 for optimal cost savings. Example 4: Device capable of detecting Tion OPs and / or OPs with low Ki.

[0116] As observed, in insects and mammals, cytochrome c P450 in the liver (in the presence of NADPH) converts OPs from the ion form to the oxon form. In preliminary studies (Figures 1B and 1D), a 10-fold and even 50-fold increase in ki of rHuCE versus paration was achieved in vitro. These chemical oxidants are much more potent than P450 and should convert ions to oxons more rapidly, thus increasing the detection speed of OP / C. Similarly, the P450 assay can be optimized in the same way to optimize oxidation conditions.

[0117] For chemical oxidation, it has been shown that oxidation by iodine catalysts or Fenton reagent readily converts the parathion to paraoxon; with toxicity readily increased in AChE-based assays. This same strategy can be readily optimized in vitro and translated into device form factor to rapidly demonstrate capability with rHuCE. Such chemicals can be more potent than P450 and can greatly increase the reaction rate and color change in a device.

[0118] Reference to any prior art in this descriptive report is not, and should not be taken as, an acknowledgment or suggestion in any way that such prior art forms part of common general knowledge.

[0119] It will be evaluated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without diverging from the scope of the present invention as presented and defined by the following claims. The entire teachings of any patents, patent applications or other publications referred to in the present invention are incorporated by reference in the present invention as if Petition 870210102169, dated 05 / 11 / 2021, pp. 330 / 341 68 / 68 fully presented in the present invention. Petition 870210102169, dated 05 / 11 / 2021, pp. 331 / 341

Claims

1 / 8 CLAIMS 1. Device for detecting an OP / C compound characterized in that it comprises: (a) a top piece comprising the first carrier material, wherein said first carrier material comprises an immobilized OP / C detection enzyme; (b) a first substrate; (c) a second enzyme; (d) a second substrate; (e) a pH-sensitive dye; (f) a second carrier material; (g) an ampoule comprising a buffer; (h) an intermediate piece;and (i) a bottom piece, wherein the intermediate piece is associated with the top piece and the bottom piece, wherein the intermediate piece comprises the second carrier material and the ampoule, and wherein when the intermediate piece is rotated relative to the top piece or the bottom piece, the ampoule is capable of being broken to release the stopper to contact the first carrier material and the second carrier material causing: (i) the enzymatic conversion of the first substrate by the OP detection enzyme to produce an acidic reaction product; and (ii) the enzymatic conversion of the second substrate by the second enzyme to produce a basic reaction product; and (j) optionally an oxidant. Petition 870210102169, dated 11 / 05 / 2021, pp. 332 / 341 2 / 8; 2. Device according to claim 1, characterized in that the OP / C detection enzyme is: a) a hydrolase; b) a lipase, a phosphatase, an amylase, a cellulase, a protease, a peptidase, a urease or a deaminase; c) a carboxylesterase (CES), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), organophosphorus hydrolase or organophosphorus acid anhydrolase; d) CES1 or CES2; e) wild-type carboxylesterase aE7 from the Australian blowfly Lucilia cuprina (LcαE7); f) mutant form of LcaE7G137D; g) mutants of LcαE7 E183, K275, E78 and / or E292; h) wild-type AChE; i) AChE mutant, such as rHuAChE containing two mutations in the acyl pocket residues (F295L, F297V); j) carboxylesterase (Cqestβ2) from the mosquito Culex quinquefasciatus; k) selected from Tables 2 to 5;or l) a variant of the OP / C detection enzyme having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of the OP / C detection enzyme from (a) to (k), wherein the variant of the OP / C detection enzyme: (1) retains the ability to convert the first substrate to acetic acid; and (2) retains this ability when inhibited by an OP. Petition 870210102169, dated 11 / 05 / 2021, pp. 333 / 341 3 / 8; 3. Device according to claim 1 or 2, characterized in that the OP / C detection enzyme: (a) can detect at least 10 pg, at least 20 pg, at least 30 pg, at least 40 pg, at least 50 pg, at least 60 pg, at least 70 pg, at least 80 pg, at least 90 pg or at least 100 pg of an OP / C compound; (b) can detect between 10 and 100 pg, between 20 and 100 pg, between 30 and 100 pg, between 40 and 100 pg, between 50 and 100 pg, between 60 and 100 pg, between 70 and 100 pg, between 80 and 100 pg, between 90 and 100 pg of an OP / C compound; (c) comprises an inhibition rate constant ki of at least 103 M' ^min-1 to 108 M^-min'1, at least 104 M 1-min1 to 108 M^-min'1, at least 105 M ^min1 to 108 M^-min'1, at least 106 M ^min1 to 108 M^-min'1, or at least 107 M ^min1 to 108 M^-min'1; and / or (d) comprises an inhibition rate constant ki of 103 to 105 M'1-min'1, ki of 104 to 105 M'1-min'1, 105 to 106 M'1-min'1, 106 M^-min'1 to 107 M^-min'1 or 106 M'1-min'1 to 108 M^-min'1.

4. Device according to any one of claims 1 to 3, characterized in that the first carrier material comprises: (a) natural polymers, including but not limited to cellulose, hemicellulose, pectin, chitin, silk, lignin, starch, polypeptides, collagens, keratins, polysaccharides, nucleic acids, and / or rubbers; or (b) derivatives of natural polymers, including but not limited to methylation, carboxylation, amidation, sulfatization, hydroxylation, condensation, iodination, reduction, oxidation, esterification, alkylation and / or halogenation; and / or (c) synthetic polymers and copolymers, including but not limited to polyurethanes, thermoplastic polyurethanes, silicones, polyamides, polystyrenes, bakelite, polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, Petition 870210102169, dated 05 / 11 / 2021, page 334 / 341 4 / 8 polychloroprene and / or polyimides.

5. Device according to any one of claims 1 to 4, characterized in that the first carrier material is a sponge.

6. Device according to any one of claims 1 to 5, characterized in that the first carrier material is composed of polyurethane.

7. Device according to any one of claims 1 to 6, characterized in that the first substrate is selected from acetylcholine, acetylthiocholine, butyrylcholine, butyrylthiocholine, 4-nitrophenyl acetate, 4-nitrophenyl propionate, 4-nitrophenyl butyrate, 4-nitrophenyl valerate, 4-nitrophenyl dimethylacetate, 4-nitrophenyl trimethylacetate, 4-nitrophenyl guanidinobenzoate, or 6-nitrocoumarin.

8. Device according to any one of claims 1 to 7, characterized in that the second enzyme and the second substrate are selected from Table 6.

9. Device according to any one of claims 1 to 8, characterized in that the second enzyme is urease and the second substrate is urea.

10. Device according to any one of claims 1 to 9, characterized in that the basic reaction product is ammonia.

11. Device according to any one of claims 1 to 10, characterized in that the pH-sensitive dye is selected from nitrazine, phenol red, chlorophenol red, bromocresol green, cresol red, bromomethyl blue, or bromocresol violet.

12. Device according to any one of claims 1 to 11, characterized in that the device further comprises an oxidant that converts an inactive OP / C compound to an active OP / C compound. Petition 870210102169, dated 05 / 11 / 2021, pp. 335 / 341 5 / 8 13. Device according to claim 12, characterized in that the oxidant is a P450 enzyme in the presence of the cofactor NADPH.

14. Device according to claim 13, characterized in that the P450 enzyme is a wild type or a triple mutant of CYP1A2 (P450 BM-3 (CYP102-A1).

15. Device according to any one of claims 1 to 14, characterized in that: a) the first carrier material further comprises the pH-sensitive dye, the second enzyme and / or the oxidant; b) the ampoule further comprises the pH-sensitive dye; and / or c) the second carrier material comprises the pH-sensitive dye, the first substrate, the second substrate and / or the oxidant.

16. Device according to any one of claims 1 to 15, characterized in that the second carrier material is selected from: (a) natural polymers, including but not limited to cellulose, hemicellulose, pectin, chitin, silk, lignin, starch, polypeptides, collagens, keratins, polysaccharides, nucleic acids, and / or rubbers; or (b) derivatives of natural polymers, including but not limited to methylation, carboxylation, amidation, sulfatization, hydroxylation, condensation, iodination, reduction, oxidation, esterification, alkylation, and / or halogenation; and / or (c) synthetic polymers and copolymers, including but not limited to polyurethanes, thermoplastic polyurethanes, silicones, polyamides, polystyrenes, bakelite, polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polychloroprene, and / or polyimides.

17. Device according to any one of claims 1 to 16, characterized in that the pH-sensitive dye, the first substrate, the second substrate and / or the oxidant are lyophilized as a microtablet.

18. Device according to any one of claims 1 to 17, characterized in that the top piece and the intermediate piece are connected.

19. Device according to any one of claims 1 to 18, characterized in that the bulb extends into the bottom piece.

20. Device according to claim 19, characterized in that the intermediate piece contains one or more holes to allow the flow of contents released from the ampoule between the bottom piece and the intermediate piece.

21. Device according to any one of claims 1 to 20, characterized in that the device further comprises a cover.

22. Device according to claim 21, characterized in that the cover is transparent and / or comprises a window.

23. Device according to any one of claims 1 to 22, characterized in that the device comprises at least one O-ring.

24. Device according to any one of claims 1 to 23, characterized in that the device is operationally associated with a smartphone.

25. Device according to any one of claims 1 to 24, characterized in that the OP / C detection enzyme is produced by a plant cell, a mammalian cell, or a bacterial cell.

26. Method for detecting an OP / C, characterized in that it comprises: (a) contacting the device defined in any of claims 1 to 25 with a surface; (b) rotating the intermediate piece relative to the top piece or bottom piece, thereby breaking the ampoule to release the buffer to contact the first carrier material and the second carrier material, causing the enzymatic conversion of a second substrate by a second enzyme to produce ammonia; and wherein: (1) in the absence of an OP / C, the enzymatic conversion of the first substrate by the OP / C detection enzyme occurs, resulting in the maintenance of a reference pH; or (2) in the presence of an OP / C, the enzymatic conversion of the first substrate by the OP / C detection enzyme is inhibited by the OP / C compound, resulting in an increase in pH above the reference pH due to the production of the basic reaction product.

27. Method according to claim 26, characterized in that the OP / C compound is selected from: (a) an insecticide selected from: acephate, aldicarb (Temik), carbachol, carbamate, carbaryl (Sevin), carbofuran (Furadan), carisoprodol, chlorfenvinphos, chlorpyrifos-oxon, chlorpyrifos, dementon-S, diazoxon, diazinon, dichlorvos, dicrotophos, dimethoate, dithiocarbamates, EA-3990, eserine, ethienocarb, etoprophos, ethylcarbamate, felbamate, fenobucarb, fenamifos, isocarbophos, malathion, mebutamate, meprobamate, metamidaphos, methomyl, methylcarbamate, methyl parathion, methyl-POX, monocrotophos, naled, neostigmine, omethoate, oxamyl, paraoxon, parathion, phorate, phosmet, phosphamidon, rivastigmine, T-1123, terbufos, tetrachlorvinphos, tetriso, thiocarbamates (e.g., O-thiocarbamate or S-thiocarbamates), triazophos, and / or tibamate;(b) a G agent, such as Tabun (GA), Sarin (GB), Clorsarin (GC), Soman (GD), methylsarin, n-butylsarin, iso-butylsarin, n-propylsarin, ethylsarin (GE), and / or cyclosarin (GF), GV; (c) a V agent, such as EA-3148, VE, VG, VM, VP, VR, VS and / or VX; and / or Petition 870210102169, dated 11 / 05 / 2021, p. 338 / 341 8 / 8 (d) a Novichok agent, such as A-234.; 28. Method according to claim 26 or 27, characterized in that the device: (a) can detect at least 10 pg, at least 20 pg, at least 30 pg, at least 40 pg, at least 50 pg, at least 60 pg, at least 70 pg, at least 80 pg, at least 90 pg or at least 100 pg of an OP / C compound; and / or (b) can detect between 10 and 100 pg, between 20 and 100 pg, between 30 and 100 pg, between 40 and 100 pg, between 50 and 100 pg, between 60 and 100 pg, between 70 and 100 pg, between 80 and 100 pg, between 90 and 100 pg of an OP / C compound.

29. Method according to any one of claims 26 to 28, characterized in that the surface comprises food, clothing, or machinery. Petition 870210102169, dated 05 / 11 / 2021, pp. 339 / 341

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