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Issue 15, 2011
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Non-linear and linear enhancement of enzymatic reaction kinetics using a biomolecule concentrator

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Abstract

In this work we investigate concentration-enhanced enzyme activity assays in nanofluidic biomolecule concentrator chips which can be used to detect and study very low abundance enzymes from cell lysates and other low volume, low concentration samples. A mathematical model is developed for a mode of operation of the assay (J. H. Lee, B. D. Cosgrove, D. A. Lauffenburger and J. Han, J. Am. Chem. Soc., 2009, 131, 10340–10341) in which enzyme and substrate are concentrated together into a plug on chip which results in a non-linear enhancement of the reaction rate. Two reaction phases, an initial quadratic enzyme-limited phase and a later, linear substrate-limited phase, are predicted and then verified with experiments. It is determined that, in most practical situations, the reaction eventually enters a substrate-limited phase, therefore mitigating the concern for non-specific reactions of biosensor substrates with off-target enzymes in such assays. We also use this mode to demonstrate a multiplexed concentration-enhanced enzyme activity assay. We then propose and demonstrate a new device and mode of operation, in which only the enzyme is concentrated and then mixed with a fixed amount of substrate in an adjacent picolitre-scale reaction chamber. This mode results in a linear enhancement of the reaction rate and can be used to perform mechanistic studies on low abundance enzymes after concentrating them into a plug on chip.

Graphical abstract: Non-linear and linear enhancement of enzymatic reaction kinetics using a biomolecule concentrator

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Supplementary files

Article information


Submitted
11 Nov 2010
Accepted
06 May 2011
First published
16 Jun 2011

Lab Chip, 2011,11, 2569-2576
Article type
Paper

Non-linear and linear enhancement of enzymatic reaction kinetics using a biomolecule concentrator

A. Sarkar and J. Han, Lab Chip, 2011, 11, 2569
DOI: 10.1039/C0LC00588F

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