An immobilization multienzyme microfluidic chip for acetylcholinesterase inhibition assay by fluorescence method

Abstract

A bi-enzyme immobilized microfluidic device was developed for the rapid enzyme inhibition assay by fluorescence detection. The micro-channels of the chip were activated by using an acidic hydrogen peroxide (H₂O₂) solution, followed by amination with a 3-aminopropyltriethoxysilane/methyltriethoxysilane (APTES/MTES) mixture, and then followed by glutaraldehyde grafting and subsequent covalent protein binding. The analysis was based on the oxidation of L-tyrosine to its tyrosine dimer in the presence of horseradish peroxidase (HRP) and H₂O₂, which has maximal excitation and emission wavelengths at 320 nm and 416 nm, respectively. H₂O₂ was an end product under the catalysis of acetylcholinesterase (AChE) and choline oxidase (ChO) immobilized on the inner surface of a microchip, using acetylcholine (ACh) as the enzyme substrate. Fluorescence of the dimer was directly proportional to the concentration of the H₂O₂ and partly reflected enzyme activity. Thus, AChE inhibition activity was indicated by a decrease in fluorescence signal detected by a microplate reader. The effects of flow velocities, reaction time and buffer systems (pH, types and concentration) on the reaction system were investigated. The utility of the developed microchip was demonstrated for AChE inhibition studies using donepezil as a model compound, which relied upon the generation of a uniform concentration of substrate and a microfluidically created concentration gradient of inhibitor using a single initial inhibitor concentration. The IC₅₀ value of donepezil was determined and found to correlate well with that obtained in the microplate. Accordingly, the microchip can be applied to the screening of enzyme inhibitor and enzymatic reaction kinetics study, which may be useful for drug discovery and screening.