Badges of Immobilized Enzymes: Detection of Chemical Warfare Agents

Rapid detection of chemical warfare agents (CWAs) in all of its forms including gas and liquid is of paramount importance to protect the warfighter or first responders in homeland defense. CWAs such as OPs and vesicants pose serious threats because these agents can be ingested with contaminated food or rapidly penetrate skin. We reported that polyurethane immobilized cholinesterases (acetyl and butyryl) yielded a product with similar enzyme activity as soluble enzyme. While enzyme sensors have the advantage of selectivity, sensitivity and, most important, specificity, ChEs are particularly ideal enzymes for rapid detection of OPs because they possess some of the fastest turnover rates known for any enzyme, and are the most sensitive targets for CWAs including GA (tabun), GB (sarin, the agent used by the terrorist group in Japan in 1995), GD (soman), and VX. We also reported that immobilized cholinesterase biosensors exhibited remarkable stability suitable for detection of OPs in both air and water, and stability to harsh environmental conditions because the immobilized enzymes do not leach from the polyurethane support. We now describe two improvements over current OP detecting kits. (1) Immobilized enzyme badges are being designed as a field system capable of identifying the type of OP present. This will aid in treatment. (2) A coupled enzyme reaction also provides rapid colorimetric or electrochemical indication of vesicants (HD). Therefore, one badge detects two classes of CWAs. With the constant threat of chemical warfare or terrorist acts, the development of alternative means to rapidly identify CWAs is critical. INTRODUCTION Enzyme sensors have the advantage of selectivity, sensitivity and, most important, specificity, ease and portability, and markedly simplified instrumentation. Biosensors based on cholinesterases (ChEs) immobilized non-covalently have been prepared by a variety of processes. The currently fielded spot M256A1 chemical agent detector kit and the M272 water test kit use dry eel ChE non-covalently applied onto fiber or ion-exchange paper. It can only be exposed to air/vapor environmental conditions. An immobilized enzyme will not leach from the polyurethane support so that the product an OP badge can now be used to sample anything from soil, water, to air. Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 01 JUL 2003 2. REPORT TYPE N/A 3. DATES COVERED 4. TITLE AND SUBTITLE Badges Of Immobilized Enzymes: Detection Of Chemical Warfare Agents 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Walter Reed Army Institute of Research, Division of Biochemistry, Department of Biochemical Pharmacology 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES See also ADM001523. 14. ABSTRACT 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT UU 18. NUMBER OF PAGES 9 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 It has been demonstrated that a variety of enzymes exhibited enhanced mechanical and chemical stability when immobilized on solid supports, thus producing a biocatalyst. The study of degradation of organophosphates by immobilized enzymes dates back to Munnecke, who attempted to immobilize a pesticide detoxification extract from bacteria by absorption on glass beads. The absorbed extract retained activity for a full day. Wood and coworkers, using isocyanate-based polyurethane foams (Hypol®), found that a number of different enzymes could be covalently bound to this polymer and retain their activity; after that Havens and Rase successfully immobilized parathion hydrolase. METHODS ChE N H C N H R" O R' N H C N H R" O ChE-NH2 + R"NCO R'NH2 + R"NCO R' N H C OH O R'NH2 + CO2 (gas) R' N H C OH O R'NCO + H2O