Chemiluminescence-based pesticide biosensor utilizing the intelligent evolved properties of the enzyme alkaline phosphatase
A methodology is described for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer, poly(3-undecylthiophene-co-3- methanoithiophene). A streptavidin conjugate of alkaline phosphatase is used in this study. The biotinylated polymer is attached to the silanized glass surface via hydrophobic interactions and the enzyme is interfaced with the polymer through the classical biotin- streptavidin interaction. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro) (1,2 dioxetane-3-2`-tricyclo-) (3.3.1.1 )-(decani-4-yl) phenyl phosphate, to a species which emits energy by chemiluminescence. This chemiluminescence signal can be detected with a photomultiplier tube for enzymatic catalysis with the biocatalyst both in solution and immobilized on a glass surface. The signal generation is inhibited by the organophosphorus based insecticides such as paraoxon as well as nerve agents. We demonstrate in this study that a number of organophosphorus based insecticides inhibit the enzyme-mediated generation of chemiluminescence signal. This is true for the enzyme conjugate both free in solution and immobilized on a glass surface. In solution, the inhibition resembles the case of a partially uncompetitive system. By this type of inhibition we are able to detect pesticides down to about 50 ppb for the enzyme in solution. The pesticide detection limit of immobilized enzyme is currently being investigated. The enzyme is capable of a number of measurement cycles without significant loss of signal level.
- Research Organization:
- Massachusetts Univ., Lowell, MA (United States)
- OSTI ID:
- 147357
- Report Number(s):
- AD-A-295958/3/XAB; CNN: Contract DAAL03-91-G-0064; TRN: 53240028
- Resource Relation:
- Other Information: PBD: 31 Dec 1994
- Country of Publication:
- United States
- Language:
- English
Similar Records
Imaging of DNA sequences with chemiluminescence
Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry