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

Søren Jepsen,Thomas Jørgensen,Henrik Sørensen,Søren Kristensen

doi:10.3390/s19030539

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, KM 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.

Highlights

The detection and quantification of enzymatic reactions is widely used in many areas of industrial production and biomedical assays
We examined if the observed change in refractive index (RI) during the initial reaction rate was suitable for determining the kinetic Michaelis–Menten constant (KM )
Our results index sensing can be used in the estimation of enzyme kinetics, the system should be further demonstrated that refractive index sensing can be used in the estimation of enzyme kinetics, optimized to handle low substrate concentrations

Summary

Introduction

The detection and quantification of enzymatic reactions is widely used in many areas of industrial production and biomedical assays. There is a wide range of well-established methods and sensors for measuring such reactions, including but far from limited to colorometric, polariometric and amperometric sensors [1] Many of these sensors rely on a coupled reaction scheme with secondary reactants that form a detectable biproduct, e.g., NAD+ /NADH, or enable a transfer of electrons to electrodes to produce a detectable signal. The implementation of such sensors into miniaturized microfluidic systems is challenged by the need to include additional reactants and to ensure the precise mixing of sample and reactant. Direct detection can be possible if either the substrate or the product exert distinct physiochemical properties such as chirality, intrinsic fluorescence or in this case, a different refractive index (RI)

Methods

Results

Discussion

Conclusion