Real-time interferometric refractive index change measurement for the direct detection of enzymatic reactions and the determination of enzyme kinetics

Søren T. Jepsen*, Thomas M. Jørgensen, Henrik S. Sørensen, Søren R. Kristensen

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.

Original languageEnglish
Article number539
JournalSensors (Switzerland)
Volume19
Issue number3
Number of pages8
ISSN1424-8220
DOIs
Publication statusPublished - 2019

Keywords

  • Dn/dc
  • Enzyme
  • Interferometry
  • Refractive index

Cite this

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title = "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.",
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author = "Jepsen, {S{\o}ren T.} and J{\o}rgensen, {Thomas M.} and S{\o}rensen, {Henrik S.} and Kristensen, {S{\o}ren R.}",
year = "2019",
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language = "English",
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journal = "Sensors",
issn = "1424-8220",
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}

Real-time interferometric refractive index change measurement for the direct detection of enzymatic reactions and the determination of enzyme kinetics. / Jepsen, Søren T.; Jørgensen, Thomas M.; Sørensen, Henrik S.; Kristensen, Søren R.

In: Sensors (Switzerland), Vol. 19, No. 3, 539, 2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Real-time interferometric refractive index change measurement for the direct detection of enzymatic reactions and the determination of enzyme kinetics

AU - Jepsen, Søren T.

AU - Jørgensen, Thomas M.

AU - Sørensen, Henrik S.

AU - Kristensen, Søren R.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Dn/dc

KW - Enzyme

KW - Interferometry

KW - Refractive index

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DO - 10.3390/s19030539

M3 - Journal article

VL - 19

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JF - Sensors

SN - 1424-8220

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ER -