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    Design and synthesis of cyclodiene insecticide biosensors and fluorescent probes

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    Author
    Brummel, Kathleen Elizabeth
    Advisor
    Wright, Jeremy, Ph.D.
    Date
    1997
    Type
    dissertation
    
    Metadata
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    Abstract
    A fiber optic evanescent fluorosensor has been developed for the detection of cyclodiene insecticides which possess hexachlorobicyclic ring systems. Four polyclonal (pAb) and one monoclonal (mAb) antibodies have been generated from synthesized haptens by conjugation with bovine serum albumin (BSA). These include, 6-(1,4,5,6,7,7-hexachloro-bicyclo(2.2.1) heptane-2, 3b-succinimido)-caproic acid (CCA), 3-(1,4,5,6,7,7-hexachloro-bicyclo(2.2.1) heptane-2, 3b-succinimido)-alanine acid (CAPA), 6,7-dihydro-6-carboxyaldrin (ALD-COOH) and N-(5-carboxypentanyl)-5-norbornene-2,3-dicarboxyimide(2,3b) bicyclo-1,4,5,6,7,7-hexachloro-cycloheptane (HC-NBCA). Their respective Abs were anti-CCA, anti-CAPA, anti-ALD, anti-HCNBCA and mAb IF11. Fluorescein conjugates of 1,4,5,6,7,7 hexachloro-5-norbornene 2,3 dicarboxylic anhydride, CCA and ALD-COOH were synthesized and designated as FL-CCA, FL-CCA long chain and FL-ALD. Antibodies immobilized covalently on quartz fibers were used to detect chlordane, heptachlor, dieldrin, endrin, aldrin and endosul fan by competitive inhibition of binding of the fluorescein conjugates (designated FL-probes). FL-ALD (10 nM) demonstrated affinity for anti-ALD, anti-HCNBCA and anti-CCA Ab-coated fibers at 50 mug/mL. In competition studies with anti-ALD, 0.1 muM aldrin and CCA inhibited fluorescence by 50% and 45%, respectively. FL-ALD was inhibited by 53% with heptachlor and 62% with aldrin at 0.1 muM on anti-HCNBCA fibers and CCA caused 73% and aldrin 40% inhibition on anti-CCA fibers. Anti-CAPA demonstrated affinity towards FL-CCA long chain with a slow dissociating component upon removal of the FL-probe from the flow buffer. FL-CCA binds to anti-CCA with high affinity (K subscript D=1.9 nM) and reversibility. Competitive inhibition of FL-CCA by the different cyclodiene insecticides showed CCA > chlordane > heptachlor > dieldrin > aldrin. Detection limits were 0.04 ng/mL, 0.4 ng/mL, 4 ng/mL, 4 ng/mL and 370 ng/mL, respectively. Since CCA was used to generate the antisera, cross-reactivity with other chlorinated hydrocarbons was expected. The level used to test cross-reactivity was 0.1 muM, representing the lowest concentration causing a significant change in fluorescence between compounds. The biosensor showed cross-reactivities for chlordane, heptachlor, dieldrin, endrin, endosulfan and aldrin to be 64, 60, 51, 20, 9 and 7, respectively, where CCA = 100. These experiments show that the biosensor can recognize various chlorinated insecticides based upon their structural similarities to the hapten and the fluorescein conjugates used. Extension of the research could ultimately result in an on-site detection system for cyclodiene insecticides.
    Description
    University of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 1997
    Keyword
    Chemistry, Analytical
    Chemistry, Pharmaceutical
    Chemistry, Agricultural
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1407
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    Theses and Dissertations All Schools
    Theses and Dissertations School of Pharmacy

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