Enzyme kinetic measurements using fluorometric spectroscopy in a microfluidic chips.

摘要

Microfluidics is an emerging multidisciplinary field that combines physics, chemistry, engineering, microtechnology and biotechnology with the purpose of designing of systems that will be using miniature amounts of fluids. There are many benefits to miniaturization such as low consumption of reagents, and high-throughput fabrication of devices, lower costs, and more accuracy in measurement in most cases.;In this study we have demonstrated a rapid, quantitative analysis method for enzyme kinetics measurement of horseradish peroxidase (HRP) using fabricated microchannels florescence spectroscopy at physiological pH. This methodology provides an alternative to kinetic measurements using traditional enzyme assays with advantages like process miniaturization, real-time monitoring capability and integration into droplet-based assays. Our goal was achieved by measuring the reaction rate of the substrate (H2O2) at various concentrations by estimating the rate of formation of the product (resorufin), which has an absorption/excitation and emission wavelength at 560 nm and 590 nm respectively. The rate of formation of product was product was measured in two platforms, in order to compare the results, first using a UV-Vis spectrophotometer and then using our microchips. By using the Michaelis-Menten equation and nonlinear regression we were able to estimate the Km value of HRP in both platforms to be 116muM (at 560 nm for fluorescence) and 150muM (at 590 nm for absorbance), which were reasonably consistent with other published literatures (84 +/- 13muM).