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Title:Chemiluminescence detection of NADH and enzyme substrates with immobilized tris(2,2'-bipyridyl)ruthenium (II) and dehydrogenase enzymes
Author(s):Martin, Alice Fay
Doctoral Committee Chair(s):Nieman, Timothy A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Analytical
Chemistry, Biochemistry
Abstract:The objective of this work is to couple the sensitivity of tris(2,2$\sp\prime$-bipyridyl)ruthenium(II) (Ru(bpy)$\sb3\sp{2+}$) electrogenerated chemiluminescence (ECL) to the selectivity of dehydrogenase enzymes. Within this objective, methods of reagent immobilization for Ru(bpy)$\sb3\sp{2+}$ and dehydrogenase enzyme have been studied and applied to bioanalytical determinations. Dehydrogenase enzymes employ NAD$\sp+$ as their cofactor and produce NADH in an amount equivalent to reacted analyte. Detection of NADH via the ECL reaction with Ru(bpy)$\sb3\sp{3+}$ is then used to determine analyte concentration.
Several different materials have been investigated as immobilization matrices. Sulfonic cation exchange polymers Nafion (Dupont) and AQ55 (Eastman) have been used for Ru(bpy)$\sb3\sp{2+}$ immobilization to create ECL sensors capable of detecting 5 $\mu$M NADH and lower. The ECL sensor was created by exchanging Ru(bpy)$\sb3\sp{2+}$ into a film of Nafion or AQ55 cast on a platinum electrode. The electrode was then used to generate Ru(bpy)$\sb3\sp{3+}$ which then is available to react with NADH to yield light.
Dehydrogenase immobilization has been carried out in two different formats. First, an enzyme reactor was prepared by covalently crosslinking the dehydrogenase to amino-derivatized glass beads and packing them in a column. The column was then used on-line in a flow injection analysis system with the ECL sensor as a detector. The conditions for optimum ECL signal were: pH (about 6.5), flow rate (2 mL/min), and NAD$\sp{+}$ concentration (1-2.5 mM). Glucose detection was from 5.0 mM to 10 $\sb\mu$M glucose.
The second format entrapped the dehydrogenase in AQ55 to be used in an ECL biosensor. Glucose, L-lactate or alcohol dehydrogenase were mixed with AQ55(D) to form an enzyme layer on top of or near a polymer modified electrode containing Ru(bpy)$\sb3\sp{3+}.$ The enzyme layer was treated with an overlayer of Nafion to protect the polymer film from redissolving in an aqueous solution. Three biosensor designs based on different physical positions of the two layers were evaluated. Using a glucose dehydrogenase based sensor, glucose is detected from 3.0 mM to 10 $\mu$M. Additionally, it has been shown that both NAD$\sp+$ and NADP$\sp+$ may be used as dehydrogenase cofactors where NADPH itself may be detected from 10 nM to 100 $\mu$M.
Issue Date:1995
Rights Information:Copyright 1995 Martin, Alice Fay
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9543667
OCLC Identifier:(UMI)AAI9543667

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