Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics

Abstract

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, K_M was found to be 0.33 mM using Michaelis⁻Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Keywords: dn/dc; enzyme; interferometry; refractive index.

Figure 1

The enzymatic phosphorylation of glucose to glucose-6-phosphate by hexokinase, measured using BSI. (a) The real-time measurement of four glucose solutions (0.4, 1.0, 1.6 and 2.0 mM). (b) The end-point values in refractive index units vs. initial glucose concentration. Linear fit (R² > 0.99, slope 0.99 × 10⁻⁵ RIU/mM). Figure 1a is reproduced from reference [8] with permission from Elsevier.

Figure 2

Hexokinase substrate saturation curve: Initial reaction velocity (rad/min) plotted against initial glucose concentrations (mM). Red line shows non-linear regression (R² = 0.93) to the parameters in Equation 3 from which K_M is obtained.

Figure 3

The enzymatic hydrolysis of ATP and ADP to AMP by apyrase. (a) The real-time measurement of 1.6 mM ATP and ADP (duplicate runs are shown for both ATP and ADP). (b) The linear fit of end-point values in refractive index units vs. initial substrate concentration. ATP (blue) R² > 0.99, slope 1.51 × 10⁻⁵ RIU/mM, ADP (green) R² > 0.99, slope 0.68 × 10⁻⁵ RIU/mM.