Dual-enzyme, co-immobilized capillary microreactor combined with substrate recycling for high-sensitive glutamate determination based on CE.

ABSTRACT

A new method for high-sensitive determination of glutamate was developed and evaluated based on CE by using dual-enzyme co-immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co-immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet-visible spectroscopy, and fluorescence spectroscopy. The GDH-GPT co-immobilized capillary microreactor showed great stability and reproducibility. The apparent K(m) for glutamate with GDH-GPT coupled reaction was determined to be 0.61+/-0.06 mM but 2.56+/-0.24 mM when only GDH was immobilized. Glutamate determination was based on on-column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co-immobilized GDH-GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7-fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 muM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.