Issue 14, 2013
From the journal:

Lab on a Chip

Protein–protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection†

Christian Benz,ab   Heiko Retzbach,a   Stefan Nagla  and  Detlev Belder*a  

* Corresponding authors

a Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, Leipzig, Germany
E-mail: belder@uni-leipzig.de
Fax: +49 341 97–36115
Tel: +49 341 97 36091

b LIFE–Leipzig Research Center for Civilization Diseases, Universität Leipzig, Philipp-Rosenthal-Straße 27, Leipzig, Germany

Abstract

Herein, we demonstrate the feasibility of a protein–protein 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 protein–protein 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

About
Cited by
Related
Download options Please wait...

Supplementary files

Article information

DOI
https://doi.org/10.1039/C3LC00057E
Article type
Paper
Submitted
11 Jan 2013
Accepted
15 Apr 2013
First published
16 Apr 2013

Lab Chip, 2013,13, 2808-2814

Permissions

Request permissions

Protein–protein 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

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements