Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing

Rajiv Pande; S. Kamtekar; Madhu Sudhan Rao Ayyagari; Kenneth A. Marx; Jayant Kumar; Sukant K. Tripathy; David L. Kaplan

doi:10.1117/12.209798

22 May 1995 Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements

Rajiv Pande, S. Kamtekar, Madhu Sudhan Rao Ayyagari, Kenneth A. Marx, Jayant Kumar, Sukant K. Tripathy, David L. Kaplan

Author Affiliations +

Proceedings Volume 2441, Smart Structures and Materials 1995: Smart Materials; (1995) https://doi.org/10.1117/12.209798
Event: Smart Structures and Materials '95, 1995, San Diego, CA, United States

Abstract

We describe a methodology for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer poly (3-undecylthiophene-co-3- thiophenecarboxaldehyde) 6-biotinamido hexanohydrazide attached hydrophobically to silanized glass. The biotin-streptavidin protein interaction is used to carry out this immobilization. Alkaline phosphatase catalyzes the dephosphorylation of a class of macrocyclic compounds: including CSPD {chloro 3-[4-methoxy spiro(1,2 dioxetane-3-2-trichloro-(3.3.1.1)-decan]-4 yl}phenyl phosphate to a product species which emits energy by chemiluminescence. We can detect this chemiluminescence signal with a photomultiplier tube for both enzymatic catalysis in solution and the surface immobilized enzyme (streptavidin conjugate). This enzyme is inhibited by the organophosphorus class of pesticides as well as nerve agents. The enzyme is also inhibited by Be(II), Bi(III) as well as excess Zn(II), while the apoenzyme is reactivated by Zn(II). We demonstrate in this study that two representative organophosphorus pesticides inhibit the enzymatic production of chemiluminescent products. This is true for the enzyme conjugate both free in solution and immobilized. We can detect pesticides down to about 50 ppb for the enzyme in solution and 500 ppb for surface immobilized enzyme in a 100 (mu) l capillary. Detection of Zn(II) by apoenzyme reactivation occurs down to 3 ppb. Be(II) and Bi(III) are detected by inhibition down to 1 ppm.

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Rajiv Pande, S. Kamtekar, Madhu Sudhan Rao Ayyagari, Kenneth A. Marx, Jayant Kumar, Sukant K. Tripathy, and David L. Kaplan "Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements", Proc. SPIE 2441, Smart Structures and Materials 1995: Smart Materials, (22 May 1995); https://doi.org/10.1117/12.209798

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