US 2010.004 1077A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0041077 A1 Nagy et al. (43) Pub. Date: Feb. 18, 2010 (54) PESTICIDE BIOMARKER Related U.S. Application Data (60) Provisional application No. 60/858,849, filed on Nov. (76) Inventors: Jon Owen Nagy, Missoula, MT 13, 2006. (US); Charles Mark Thompson, Missoula, MT (US) Publication Classification (51) Int. Cl. GOIN 33/545 (2006.01) Correspondence Address: GOIN 33/00 (2006.01) Angelo Castellino CI2M I/34 (2006.01) 5018 Merrimac Court C40B 40/10 (2006.01) San Diego, CA 92.117 (US) GOIN 2L/00 (2006.01) (52) U.S. Cl. ....... 435/7.92; 435/287.2:506/18: 436/531; (21) Appl. No.: 12/514,797 422/57 (57) ABSTRACT (22) PCT Filed: Nov. 14, 2007 Provided are methods, compositions and articles of manufac ture for detecting biomarkers indicative of exposure of a (86). PCT No.: PCT/US07/23954 mammal to organophosphate compounds. The interaction of such a biomarker with a receptor bound to a biopolymer S371 (c)(1), results in an optical readout that reports the presence of the (2), (4) Date: May 13, 2009 biomarker. 1A Biomarker tabun-inhibited AChE fragment Anti-OP-AChE antibody & R o: COH HN ro (CH)i" - w 1) Apply \ 2 (CH2) c/N-C e Biomarker (CH2 C % C . CO2 e N. 1 -v-c (CH2) 2) Irradiate (als C (CH2), -, \ (CH2)11 Patent Application Publication Feb. 18, 2010 Sheet 1 of 2 US 2010/004 1077 A1 FIGURE 1 1A TFGE N S Biomarker tabun-inhibited AChE fragment Anti-OP-AChE S antibody . o: HNo (CH2) w gohV we, / , \ c?, Biomarker). Apply (CH2s C % c1 . --- 92 e? N. 1 -\ -cr (CH2) 2) irradiate (R- (CH2)41 a (CH2)1 CH CH CH Sayi PA is his it Patent Application Publication Feb. 18, 2010 Sheet 2 of 2 US 2010/004 1077 A1 FIGURE 2 Planning Stages to an OP-AChE Conjugate Thin Film Detector Stages for construction of a OPAChE sensor element Protein and Conjugate and Agent Controls Peptide Controls dentification and Selection of Target Protein(s) centification Design and and Selection of GE Identification and OP-Conjugates Selection of OP Peptide Models Chemical Agents Generate Generate Monoclona Polyclonal Antibodies Antibodies Attach whole “ss Antibody Western "SAMPLE andlor Fab | Blot & ELISA OPAChE - fragments to Analyses conjugate in-f thin film ---- / ifluorogenic Diagram of a prototype device for measuring fluorogenic thin film output OP-AChE Fluorogenic Polymer thin film slide 2 2.2 ( | Reader """""" Output Control Electronics: Read Head: (drive interface, data analysis (Sample docking port, microprocessor, stimulation source LED, user interface) & emission detector) US 2010/004 1077 A1 Feb. 18, 2010 PESTICIDE BOMARKER processing, as shown in the conversion 1a (thionate) to 1b (P—O, Oxon) to become active and toxic whereas reactive CROSS-REFERENCE TO RELATED organophosphoryl compounds represented by structure (2) APPLICATIONS are in the oxon form and are directly toxic. The oxon forms 0001. This application is a national phase application (1b & 2) are primarily responsible for their neurotoxic under 35 USC S371 of international patent application no. mechanism of action (described further below). PCT/US07/23954 filed Nov. 14, 2007, which claims priority 0005 Between 150,000 and 300,000 OP-related toxicity from U.S. provisional application No. 60/858,849 filed on incidences are reported yearly in the US (Rosenstock, 1991) Nov. 13, 2006, both of which are incorporated by reference in and several million people are their entireties. FIELD OF THE INVENTION S O 0002. The invention relates to detection of a biomarker RO- 7 He derived from interaction of a polypeptide with a compound or OR a metabolite thereofthat covalently modifies the polypeptide. 1a: phosphorothionate A particular embodiment disclosed is the detection of a biom insecticide arker resulting from an interaction of a serine hydrolase. Such R = Me, Et; Z = leaving group as acetylcholinesterase, with a organophosphoryl pesticide or Q O a reactive organophosphoryl compound using an optical sen : | sor incorporating an antibody that recognizes the biomarker RO- 7 X- - Z. and is immobilized onto a biopolymer material which under goes a change in an optical property upon binding of the ÖR : Y biomarker. Additionally disclosed are biosensor devices and 1b: oxon 2: highly reactive optical sensor modules that are used or incorporated into organaophosphoryl these devices for biomarker detection. BACKGROUND OF THE INVENTION 0003. The invention methods and devices for detection of a biomarker derived from interaction of a polypeptide with a compound or a metabolite thereof that covalently modifies treated worldwide for exposure to OP insecticides. Owing to the polypeptide. A particular embodiment disclosed is the the mode of inhalation and toxic neurochemical mechanism detection of a biomarker resulting from an interaction of a of action, individuals at high risk to OP exposure include serine hydrolase. Such as acetylcholinesterase, with a orga children and seniors, those with airway disabilities like nophosphoryl pesticide or a reactive organophosphoryl com asthma, neurological diseases, and/or mental illness. Sub pound using an optical sensor incorporating an antibody that populations may be a greater risk owing to prior exposure to recognizes the biomarker and is immobilized onto a biopoly OPagents, such as farmers, agrochemical workers, applica mer material which undergoes a change in an optical property tors and other occupations that handle OP compounds. upon binding of the biomarker. Additionally disclosed are Between 1993 and 1996, about 65,000 cases of OP poisoning biosensor devices and optical sensor modules that are used or were reported to the US Poison Control Center and of these, incorporated into these devices for biomarker detection. 25,000 incidents involved children under age six. It is esti 0004. In a particular embodiment of the invention a biom mated that more than 1 million children age five and under (1 arker results from covalent modification of a serine hydrolase in 20) consume an unsafe dose of OPs in the US (Goldman, after interaction of the enzyme with a suicide inhibitor. In 2000). more particular embodiments the biomarker results from 0006. The devices and methods described herein address interaction of a serine hydrolase such as an acetylcholine particular needs relating to OP exposure, which includes esterase with an organophosphate compound or a metabolite monitoring for chronic or acute exposure from an organo thereof, which acts as the suicide inhibitor. The term “orga phosphate compound Such as an organophosphoryl pesticide nophosphate” is used in the art to describe chemical classes of or a reactive organophosphoryl compound compounds comprising insecticides and pesticides. Such 0007. The mechanism of OP action which is discussed for compounds are capable of modifying proteins and polypep an acetylcholinesterase applies to other cholinesterases Such tides, including cholinesterases, either directly or after acti as butyryl-cholinesterase and other proteins which provide Vation by one or more metabolic processes. Organophospho biomarkers for OP exposure and is therefore not meant to be ryl (OP) insecticides are organophosphate compounds, which limiting of the inventions disclosed herein to a particular include malathion, diazinon, chlorpyriphos and others cholinesterase or protein. The key event in the mechanism of (shown in Table 1), are the most widely used agrochemicals OP poisoning is the reaction of the OP compound with AChE for the control of insect pests in the world and represents an to afford structurally unique products that represent mecha exposure route for such environmental toxins to field work nistically precise biomarkers of exposure. Henceforth, the ers. OP insecticides in structure 1a (see below) represents one term OP-AChE conjugates refers to the initially formed OP class of organophosphoryl pesticides (organophoshothionate conjugate from OP reaction with an acetylcholinesterase or a pesticide) having a P=S moiety in comparison to some catalytically competent fragment thereof (a primary organo examples of reactive organophosphoryl compounds repre phosphate biomarker) and Subsequently formed aged deriva sented by structures 1b and 2 which have a P-O moiety. tives (secondary organophosphate biomarkers) unless indi Typically, an organophosphoryl pesticides requires metabolic cated otherwise. The present disclosure provides novel US 2010/004 1077 A1 Feb. 18, 2010 method to identify OP-AChE conjugates (i.e., organophos mers. Further described is a biosensor device which uses one phate biomarkers) whose structures may be predicted from or more OP optical sensors or optical sensor modules for mechanistic considerations and represents an advance to the analyzing exposure to an OP compound and is useful for art. assessing the extent of exposure of a Subject to an OP com 0008. Therefore, a need exists for detection of organo pound to provide useful information to guide therapeutic phosphate biomarkers that is addressed by the present disclo intervention. sure which provides for detection systems and methods for assessing the amounts, type and structure of a biomarker, BRIEF DESCRIPTION OF THE DRAWINGS Such as an OP-AChE conjugate and its aged product, in order to assist with proper therapeutic intervention from exposure 0011 FIG. 1A. An optical sensor having a biomarker of a mammal to an environmental toxin Such as an OP receptor (e.g. anti OP-AchE antibody) immobilized onto a compound and to evaluate threats from widespread dissemi biopolymer material (e.g. a polydiacetylene polymer film) nation of Such toxins. 0012 FIGS. 1B and 1C.. Example of a biomarker binding to a biomarker receptor immobilized onto a PDA-biopolymer SUMMARY OF THE INVENTION film that induces a fluorescence change in the biopolymer material (250x250 um views are captured using a standard 0009 Biosensor devices and optical sensor employed in rhodamine excitation and red LP emission filter set). Such devices are described for detecting and discriminating (0013 FIG. 2A. Flow chart for construction an OP-optical bio-molecular products termed, in general, “biomarkers' sensor module.