Log-scale dose response of inhibitors on a chip

Abstract

We demonstrate the accommodation of log-scale concentration gradients of inhibitors on a single microfluidic chip with a semidirect dilution capability of reagents for the determination of the half-inhibitory concentration or IC(50). The chip provides a unique tool for hosting a wide-range of concentration gradient for studies that require an equal distribution of measuring points on a logarithmic scale. Using Matrix metalloproteinase IX and three of its inhibitors, marimastat, batimastat, and CP471474, we evaluated the IC(50) of each inhibitor with a single experiment. The present work could be applied to the systematic study of biochemical binding and inhibition processes particularly in the field of mechanistic enzymology and the pharmaceutical industry.

Figure 1

Design and operation of the microfluidic device. (a) 3D view of the chip. The chip is composed of three (3) different gradient formers (GF) with four microfluidic processors for each GF. Two extra processors are added for a positive and a negative controls resulting in 14 parallel reactions at the same time. (b) Step by step operation of single GF. Although a GF is composed of four reactors positive and negative controls are explained together.

Figure 2

Log-log scale standard curve for FAM with the scanned image of FAM concentration gradients.

Figure 3

Determination of enzyme kinetic parameters, K_M and k_cat. Comparison of on-chip and off-chip results indicates no need of inner filter effect correction factor for on-chip data. The off-chip velocities, after applying correction factor, shows marked improvement in the velocities at each substrate concentration. The error bar is obtained from three individual experiments. The exponential nature of corrector factor increases the magnitude of error bar, especially at higher substrate concentrations in corrected off-chip velocity data.

Figure 4

The logistics dose response plots for three inhibitors, marimastat, batimastat and CP471474, of MMP-9 enzyme. The dose-response plot for each inhibitor was obtained from three on-chip experiments. The IC₅₀ values for each inhibitor were calculated by curvefitting the four parameter model to the inhibitory activity data.