Protein–protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection
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.
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- Authors
- Benz, C., Retzbach, H., Nagl, S., & Belder, D.
- Affiliations
- Leipzig University (DE)
- Corresponding author
- Belder, Detlev
- Venue
- Lab on a Chip
- Publisher
- Royal Society of Chemistry (RSC)
- Published
- 2013
- Volume
- 13
- Issue
- 14
- Pages
- 2808
- ISSN
- 1473-0189 (ISSN-L)
- 1473-0197
- OpenAlex
- W2112012211
- Cited by
- 38
- References
- 48
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 1 · 2022: 2 · 2021: 6 · 2020: 1 · 2019: 4 · 2018: 2 · 2017: 6 · 2016: 3 · 2015: 4 · 2014: 4 · 2013: 3
- FWCI
- 2.57
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Lab on a Chip (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Freistaat Sachsen
- European Social Fund
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › Innovative Microfluidic and Catalytic Techniques Innovation
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Keywords
- protein-protein interaction, microfluidic droplets, fluorescence resonance energy transfer, droplet formation, avidin, streptavidin, competitive binding assay, nanomolar detection limit, fluorescence intensity ratio
- MeSH terms
- Avidin, Biotin, Equipment Design, Fluorometry, Microscopy, Fluorescence, Protein Binding, Serum Albumin, Bovine, Time Factors, Microscopy, Confocal, Streptavidin, Protein Interaction Mapping, Fluorescence Resonance Energy Transfer, Microfluidic Analytical Techniques
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