We report here on a technique to immobilize a multilayer enzyme assembly on an optic fiber surface. A multilayer of an enzyme, alkaline phosphatase, was successfully immobilized on an optical fiber surface. Chemiluminescence, ellipsometry, and surface plasmon resonance were used to characterize the structure and activity of the assembly. A chemiluminescence-based fiber optic biosensor utilizing this immobilization technique has been developed for the detection of organophosphorous-based pesticides. Detection of pesticide at sub-ppm level has been achieved for paraoxon.
Tapered fiber tips with different geometries are fabricated for developing a fiber optic biosensor. Fluorescence experiments are performed to compare the coupling efficiency of light for different fiber tip configurations. When light is generated in a "thick" layer (> 1 μm) around a fiber core, the continuously tapered tip with the steepest taper collects light more efficiently than the longer combination tapered tip. To demonstrate the applicability of our results, we have successfully detected weak chemiluminescent signal collected by a bundle of fibers with the short continuously tapered tips using a cooled CCD array detector. The chemiluminescence reaction was catalyzed by alkaline phosphatase immobilized on the fiber tips by a sol-gel technique.
We are investigating thin film and monolayer systems that involve conjugated conducting polymers and specific biological macromolecules. One class of conducting polymers, polyalkylthiophenes, are derivatized with biotin. These biotinylated polymers form the basis for a generic cassette system of attachment for any biological molecule through biotinylation or interaction with streptavidin. The high affinity of the biotin-streptavidin system, used in sequential steps, forms the basis of the cassette method. We have formed both monolayers and thin films (a few nanometers) of the cassette assembly in which phycobiliproteins are incorporated. We are investigating the optical signal transduction properties of specific phycobiliproteins (phycoerythrin, phycocyanin and allophycocyanain) using the cassette system on the inner surface of glass capillaries and on optical fiber surfaces. Phycobiliprotein photocurrent signals in conducting polymer matrices on microelectrodes are also being investigated. Our aim is to integrate the signal transduction mechanisms of the phycobiliproteins within monolayers or thin films of the conducting polymers to create biosensors and related smart materials for applications in biomedicine and biotechnology.
KEYWORDS: Polymers, Luminescence, Optical fibers, Signal detection, Proteins, Molecular self-assembly, Glasses, Chemiluminescence, Molecules, Chemical elements
We discuss the molecular self-assembly on optical fibers in which a novel method for protein attachment to the sensing tip of the fiber is used. Our objective is to assemble a conjugated polythiophene copolymer as an attachment vehicle. Subsequent attachment of the photodynamic phycobiliprotein serves as the fluorescence probe element. Following our earlier experiments from Langmuir-Blodgett deposition of these polymeric materials as thin films on glass substrates, we extended the technique to optical fibers. First, the bare fiber surface is silanized with a C18 silane compound. The copolymer (3-undecylthiophene-co-3- methanolthiophene, biotinylated at the methanol moiety) assembly on the fiber is carried out presumable through van der Waals interactions between the hydrophobic fiber surface and the undecyl alkyl chains on the polymer backbone. A conjugated Str-PE (streptavidin covalently attached to phycoerythrin) complex is then attached to the copolymer via the conventional biotin-streptavidin interaction. The conjugated polymer not only supports the protein but, in principle, may help to transduce the signal generated by phycoerythrin to the fiber. Our results from fluorescence intensity measurements proved the efficacy of this system. An improved methodology is also sought to more strongly attach the conjugated copolymer to the fiber surface, and a covalent scheme is developed to polymerize and biotinylate polythiophene in situ on the fiber surface.
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