Electrocatalytic enzyme sensors for selective and sensitive detection of biologically important molecules

Cholinergic neurons play a vital role in cognition and memory. Changes in the cholinergic system are associated with severe neurodegenerative disorders such as Alzheimer's disease (AD) and dementia. Hence, it is important to develop analytical strategies that can elucidate mechanisms of such selective cholinergic degradation and also be able to monitor the subtle changes that occur. Biomolecules that can selectively interact with disease specific biomarkers have potential applications in the development of biosensors. These biocatalysts, when integrated with conductive supports produce transducing signals leading to selective detection of the biomarkers. A key to such effective bioelectronic transduction is integration of various biointerfaces and their efficient electronic communication with the underlying conducting support.;The primary focus of the work has been the separation and quantification of cholinergic metabolites using capillary electrophoresis (CE) coupled with electrochemical detection (EC). CE allowed efficient separation of nL sample volumes while enzyme modified microelectrodes (MEs) enabled selective amperometric detection of choline (Ch) and acetylcholine (ACh). This method was used to study the rate of Ch uptake by the High Affinity Choline Uptake transporter protein (CHT) in mouse synaptosomes. The Michaelis-Menten constant of CHT was determined to be 0.79 muM. Further, a cholinomimetic bis-catechol hexamethonium analogue (DTH) was examined for its ability to selectively inhibit Ch uptake by CHT. The IC50 value of DTH was determined to be 76 muM. K I of this inhibitor determined by Dixon plots was calculated as 73 muM. DTH inhibited CHT via a mixed inhibition mode. The results obtained were in logical conclusion with established studies regarding structural aspects and affinity of CHT.;Furthermore, ME techniques were employed to develop two amperometric enzyme microsensor systems as detectors for CE for monitoring low micromolar concentrations of Ch and ACh. The first system was comprised of a trienzyme Au ME incorporating the enzymes acetylcholinesterase (AChE), choline oxidase (ChO) and horseradish peroxidase bound with a redox hydrogel polymer (HRP). Methods for enzyme immobilization onto the ME surface were studied and optimized. Efficient separation and selective amperometric detection of Ch and ACh were achieved at a low detection potential of +0.10 V vs Ag/AgCl. The high selectivity of the enzyme modified ME coupled with extraordinary sensitivity offered by CE enabled low mass detection limits of 38 amol for Ch and ACh. The method exhibited an excellent linear response from 2--2000 muM for both Ch and ACh.;An alternative approach involved integration of Prussian Blue (PB), an artificial peroxidase with AChE and ChO on Pt MEs for detection of Ch and ACh. Thermal and electrochemical deposition methods for PB were developed and optimized. PB incorporated enzyme MEs allowed detection of Ch and ACh at a low potential of -0.10 V vs Ag/AgCl. In spite of remarkably different stability conditions of the inorganic catalyst and biomacromolecules, synergistic effects between these systems were achieved to obtain excellent linearity from 10--2000 muM for Ch and ACh and operational stability of more than 60 electrophoretic runs.;The power of biomolecular catalysts was further exploited through development of a pyrroloquinoline quinone (PQQ) based glassy carbon (GC) amperometric sensor for real time detection of thiocholine (SCh). PQQ was efficiently incorporated within a conducting polypyrrole matrix that prevented its leaching from the electrode surface. PQQ effectively catalysed the oxidation of SCh and well defined current peaks with fast response times ranging from 11 to 27 s were obtained for various concentrations of hydrolyzed acetylthiocholine (ASCh). The versatility of the assay allowed real time detection of SCh, by addition of AChE into an electrolyte solution containing ASCh. In addition to real time monitoring of ASCh hydrolysis, the inhibition of this hydrolysis process was also observed by addition of carbofuran, an inhibitor of AChE. The extent of inhibition of AChE was sensitive to the amount of carbofuran allowing sensitive detection of potential neurotoxins. The versatility of this sensor seemed promising for further development of various bioassays.