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A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization
KTH, School of Biotechnology (BIO), Nano Biotechnology.ORCID iD: 0000-0001-5232-0805
KTH, School of Biotechnology (BIO), Nano Biotechnology.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Nano Biotechnology.
2012 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 3, 436-439 p.Article in journal (Refereed) Published
Abstract [en]

We present a novel homogeneous (mix-incubate-read) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP-based assay we detect streptavidin concentrations as low as 500?nM and demonstrate that an FP assay allows us to distinguish droplets containing 5?mu M rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.

Place, publisher, year, edition, pages
2012. Vol. 33, no 3, 436-439 p.
Keyword [en]
Droplet microfluidics, Fluorescence polarization, Homogeneous assays, Miniaturization, Proteins
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-30459DOI: 10.1002/elps.201100350ISI: 000299629800004ScopusID: 2-s2.0-84863075291OAI: diva2:400225
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20110225. Updated from manuscript to article in journal.

Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2013-04-15Bibliographically approved
In thesis
1. Droplet microfluidics for high throughput biological analysis
Open this publication in new window or tab >>Droplet microfluidics for high throughput biological analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many areas of biological research increasingly perform large-scale analyses.  In genomics the entire gene repertoire of an organism is analyzed.  Proteomics attempts to understand the function and expression patterns of all proteins in a cell or organism.  Cell biologists study large numbers of single cells to understand the heterogeneity of cell populations.  In biotechnology and synthetic biology researchers search for new functional biomolecules in large libraries of biomolecular diversity e.g. for uses in medicine or bioprocessing.  More and more all of these fields employ high throughput methods to achieve the scale of analysis necessary.

Miniaturization and parallelization provide routes towards high throughput analysis, which have proven successful for microelectronics as well as for DNA sequencing.  For the analysis of cells and biomolecules, native to an aqueous environment, miniaturization and parallelization hinges on the handling and parallel processing of very small amounts of water.  Droplet microfluidics utilizes stable picoliter (water) droplets contained in inert fluorinated oils as compartments in which to isolate and analyze cells, molecules or reactions.  These droplets can be manipulated, detected and analyzed at rates of thousands per second in microfluidic modules combining top-down microscale fabrication with the self-assembly of droplets of exact size.

The studies constituting this thesis involve new droplet based biomolecular and single cell assays, manipulation techniques and device fabrication methods to extend the capabilities of droplet microfluidics for high throughput biological analysis.

The first paper in the thesis describes a novel analysis method for studying the low abundant biomarkers present on the surface individual cells at resolutions not available by flow cytometry, the current gold standard of single cell analysis.  The use of a fluorescent optical dye code enabled the analysis of several single cell samples concurrently, improving throughput.

Further a deterministic lateral displacement module, providing passive separation of droplets by size in a microfluidic circuit at more than twice higher rates than previously achievable was demonstrated.  Using this module, droplets were separated for cell occupancy based on a cell induced droplet size transformation, which couples a biological property of the droplet contents to a physical property of the droplet.  This effect, which enables passive separation of at high throughput, indicates a potential novel assay format for clone selection.

One important feature of droplets for encapsulated single cell analysis is retention of secreted molecules providing a genotype-phenotype link.  With the objective of detecting antibody molecules secreted by hybridoma for selection, Paper III demonstrates the adaption of a homogeneous fluorescence polarization based, “mix-incubate-read”, assay for antibody detection.  In the final paper of the thesis the development of inexpensive and robust optical filters monolithically integrated in the microfluidic chip is reported. These defined filters enable integration of multiple optical filters in a polymer microfluidic device.

Overall, droplet microfluidics combines techniques for handling and manipulating millions of discrete biocompatible picoliter compartments per hour with dedicated assays for biomolecule and single cell analysis. The scale of analysis that this enables is certain to impact life science research.


Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 90 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2011:3
Microfluidics, Droplet microfluidics, High throughput biology, Single cell analysis, Hydrodynamic separation, Enzyme amplification, Fluorescence polarization, Microfabrication
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-30463 (URN)978-91-7415-858-8 (ISBN)
Public defence
2011-03-18, F2, Linstedtsvägen 26, KTH, Stockholm, 06:09 (English)
QC 20110225Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2011-02-25Bibliographically approved

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