(562f) Determining Ligand/Receptor Affinities with an Electrochemical Detection Scheme Based on a b-Galactosidase Conjugate
AIChE Annual Meeting
2005
2005 Annual Meeting
Sensors
Topical Sensors: Catalytic & Industrial Sensors
Thursday, November 3, 2005 - 4:55pm to 5:15pm
A novel enzyme-multiplied assay technique (EMAT) incorporating β-galactosidase (β-gal) as the reporter enzyme with electrochemical (ElectroEMAT) signal transduction provides an effective means for dilute chemical detection. Our EMAT system relies on a β-gal-ligand conjugate whose activity is dependent on the binding of the natural receptor to the covalently attached ligand. Receptor association with enzyme-bound ligand causes complete enzymatic activity repression, due presumably to steric hindrance at the enzyme's active site. Enzyme activity is de-repressed in the presence of free ligands, which compete with the enzyme-coupled ligand for receptor binding. Activity is measured through enzyme-catalyzed hydrolysis of the substrate p-aminophenyl β-D-galactopyranoside, which liberates the electrooxidizable product, p-aminophenol.
ElectroEMAT was used to monitor estrogen and endocrine disrupting chemical (EDC) binding to the human estrogen receptor (hER) with an estradiol-β-gal conjugate, as well as benzodiazepine drug affinity to the central benzodiazepine receptor (CBR) with a gidazepam-β-gal conjugate. EDCs interfere with normal operation of the endocrine system, often through mimicry of hormones. Using ElectroEMAT, we investigated the following estradiol-mimicking EDCs: nonylphenol, genistein, bisphenol A, and diethyl phthalate. Benzodiazepines enhance the inhibitory effect of the neurotransmitter, GABA, through binding to the GABAA receptor complex and are known to act as anti-convulsants, anxiolytics, myorelaxants, and hypnotics. We estimated CBR binding affinities to the following benzodiazepines using ElectroEMAT: gidazepam, lorazepam, and diazepam. This is the first reported homogeneous electrochemical receptor assay for detection of benzodiazepines. Binding affinities were derived from empirical data using a competitive binding assay model.
Our EDC findings indicate that nonylphenol and genistein (Ka~108 M-1) have a greater affinity for hER than bisphenol A (106 M-1) and diethyl phthalate (104 M-1). CBR binding affinity was determined, in decreasing affinity, to be: lorazepam (108 M-1), diazepam (107 M-1), and gidazepam (107 M-1). These results are consistent with published results and illustrate the effectiveness of an ElectroEMAT system for the simple detection of hormone mimicking compounds and for the screening of receptor-binding drugs. We currently are working on the construction of a reusable biosensing probe for convenient application of ElectroEMAT. This β-gal-based EMAT approach could be extended readily to other ligand/receptor pairs or to enzyme/inhibitor systems to enable high-throughput screening for ligand- and inhibitor-mimicking drugs.
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