Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

Ansgar Huebner; Luis F. Olguin; Daniel Bratton; Graeme Whyte; Wilhelm T. S. Huck; Andrew J. De Mello; Joshua B. Edel; Chris Abell; Florian Hollfelder

doi:10.1021/ac800338z

Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

Ansgar Huebner, Luis F. Olguin, Daniel Bratton, Graeme Whyte, Wilhelm T. S. Huck, Andrew J. De Mello, Joshua B. Edel, Chris Abell, Florian Hollfelder

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186 Citations (Scopus)

Abstract

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

Original language	English
Pages (from-to)	3890-3896
Number of pages	7
Journal	Analytical Chemistry
Volume	80
Issue number	10
DOIs	https://doi.org/10.1021/ac800338z
Publication status	Published - 15 May 2008

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10.1021/ac800338z

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abstract = "We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.",

author = "Ansgar Huebner and Olguin, {Luis F.} and Daniel Bratton and Graeme Whyte and Huck, {Wilhelm T. S.} and {De Mello}, {Andrew J.} and Edel, {Joshua B.} and Chris Abell and Florian Hollfelder",

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T1 - Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

AU - Huebner, Ansgar

AU - Olguin, Luis F.

AU - Bratton, Daniel

AU - Whyte, Graeme

AU - Huck, Wilhelm T. S.

AU - De Mello, Andrew J.

AU - Edel, Joshua B.

AU - Abell, Chris

AU - Hollfelder, Florian

PY - 2008/5/15

Y1 - 2008/5/15

N2 - We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

AB - We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

U2 - 10.1021/ac800338z

DO - 10.1021/ac800338z

M3 - Article

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SN - 0003-2700

VL - 80

SP - 3890

EP - 3896

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 10

ER -

Development of Quantitative Cell-Based Enzyme Assays in Microdroplets

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