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Issue 14, 2013
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Protein–protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection

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Abstract

Herein, we demonstrate the feasibility of a proteinprotein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of proteinprotein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

Graphical abstract: Protein–protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection

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Supplementary files

Article information


Submitted
11 Jan 2013
Accepted
15 Apr 2013
First published
16 Apr 2013

Lab Chip, 2013,13, 2808-2814
Article type
Paper

Proteinprotein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection

C. Benz, H. Retzbach, S. Nagl and D. Belder, Lab Chip, 2013, 13, 2808
DOI: 10.1039/C3LC00057E

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