| Two different types of biocatalytic electrochemical detection devices have been designed, developed, and coupled to a capillary electrophoretic separation system for the determination of thiols and choline (Ch)/acetylcholine (ACh).;For thiol determination, pyrroloquinoline quinone (PQQ) was used as a biocatalyst for thiol oxidation. PQQ was entrapped into the polypyrrole (PPy) film on a carbon electrode surface by electropolymerization of pyrrole in the presence of PQQ. PPy, which is known as a conducting polymer, serves not only as an immobilization matrix for PQQ, but also as an electron pathway between PQQ and the electrode surface. Using the PQQ/PPy-modified electrode, cysteine, homocysteine, penicillamine, N-acetylcysteine, and glutathione were quantitatively detected with concentration detection limits in the muM range at pH 3.5 and nM range at pH 8.4. Selectivity of the sensor toward thiol was increased by adjusting the detection potential and the solution pH. After miniaturization, the sensor was applied to a capillary electrophoretic (CE) separation system as an electrochemical detector. This system provided excellent separation and detection of a mixture of thiols with concentration detection limits in the nM range, which are equivalent to mass detection limits in the fmol range. Then, the amount of cysteine in two different dietary supplements and human urine samples was determined with high accuracy by employing this system.;For the electrochemical determination of ACh/Ch, a bi-polymer/bi-enzyme-modified electrode was prepared. A platinum electrode was first modified with PPy, followed by polytyramine (PTy) by electropolymerization of the corresponding monomers. PTy on the electrode has primary amine groups on its surface which were used to covalently attach the enzymes, choline oxidase and acetylcholinesterase via the bi-functional agent, glutaraldehyde. When the modified electrode was combined with CE, ACh and Ch could be separated and quantitatively detected by monitoring the oxidation of hydrogen peroxide produced from the enzymatic reactions at the electrode surface. This electrode showed simple Michaelis-Menten enzyme kinetics with the KM values of 0.92 mM and 1.6 mM for ACh and Ch, respectively. The modified electrode produced a stable response for about 50 hours of repeated use. The PPy and PTy films provided excellent selectivity due to their permselective feature which can eliminate large electroactive interferences and allow the selective detection of hydrogen peroxide, which is small enough to diffuse into these films. This separation/detection system was then used to analyze Ch uptake in mouse brain samples. |
