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Droplet microfluidics for high throughput biological analysis
KTH, School of Biotechnology (BIO), Nano Biotechnology.ORCID iD: 0000-0001-5232-0805
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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:3
Keyword [en]
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
Identifiers
URN: urn:nbn:se:kth:diva-30463ISBN: 978-91-7415-858-8 (print)OAI: oai:DiVA.org:kth-30463DiVA: diva2:400229
Public defence
2011-03-18, F2, Linstedtsvägen 26, KTH, Stockholm, 06:09 (English)
Opponent
Supervisors
Note
QC 20110225Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2011-02-25Bibliographically approved
List of papers
1. Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets
Open this publication in new window or tab >>Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets
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2009 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, no 14, 2518-2521 p.Article in journal (Refereed) Published
Abstract [en]

Finding the few: Cell-surface proteins are useful disease biomarkers, but current high-throughput methods are limited to detecting cells expressing more than several hundred proteins. Enzymatic amplification in microfluidic droplets (see picture) is a high-throughput method for detection and analysis of cell-surface biomarkers expressed at very low levels on individual human cells. Droplet optical labels allow concurrent analysis of several samples.

Keyword
droplets, enzymatic amplification, high-throughput screening, microfluidics, proteins, polymerase-chain-reaction, protein expression, picoliter droplets, single cells, devices, microdroplets, encapsulation, molecules, therapy, gene
Identifiers
urn:nbn:se:kth:diva-18305 (URN)10.1002/anie.200804326 (DOI)000264661600014 ()2-s2.0-70349782198 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Droplet size based separation by deterministic lateral displacement: separating droplets by cell-induced shrinking
Open this publication in new window or tab >>Droplet size based separation by deterministic lateral displacement: separating droplets by cell-induced shrinking
2011 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 7, 1305-1310 p.Article in journal (Refereed) Published
Abstract [en]

We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). We also show that droplets containing Saccharomyces cerevisiae shrink significantly during incubation while droplets containing only yeast media retain or slightly increase their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from 31% larger diameter droplets which were generated at the same time containing only media, present at a >40-fold excess. This demonstrates the resolving power of droplet separation by DLD and establishes that droplets can be separated for a biological property of the droplet contents discriminated by a change of the physical properties of the droplet. Thus suggesting that this technique may be used for e.g. clonal selection. The same device also separates 11 µm from 30 µm droplets at a rate of 12000 droplets per second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices.

Keyword
MICROFLUIDIC DEVICES, AQUEOUS DROPLET, AMPLIFICATION, EVOLUTION, SYSTEMS
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-30462 (URN)10.1039/c0lc00688b (DOI)000288455100017 ()21321749 (PubMedID)2-s2.0-79952676399 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20110225Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2017-12-11Bibliographically approved
3. A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization
Open this publication in new window or tab >>A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization
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.

Keyword
Droplet microfluidics, Fluorescence polarization, Homogeneous assays, Miniaturization, Proteins
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-30459 (URN)10.1002/elps.201100350 (DOI)000299629800004 ()2-s2.0-84863075291 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20110225. Updated from manuscript to article in journal.

Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2017-12-11Bibliographically approved
4. Monolithic PDMS passband filters for fluorescence detection
Open this publication in new window or tab >>Monolithic PDMS passband filters for fluorescence detection
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2010 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, no 15, 1987-1992 p.Article in journal (Refereed) Published
Abstract [en]

We present the fabrication and characteristics of monolithically integrated ink dyed poly(dimethylsiloxane) (PDMS) filters for optical sensing in disposable lab-on-a-chip. This represents a migration of auxillary functions onto the disposable chip with the goal of producing truly portable systems. Filters made from commercially available ink (Pelikan) directly mixed into PDMS oligomer without the use of any additional solvents were patterned with standard soft lithography technologies. Furthermore, a fabrication process based on capillary forces is presented allowing PDMS coloration of arbitrary shapes. Different filters of varying thickness fabricated using red, green and blue ink in four different concentrations were characterized. The optimal performance was found with filter thicknesses of 250 mm and ink to PDMS ratios of 0.1 (mL ink : mL PDMS oligomer) resulting in a transmittance ranging from -15.1 dB to -12.3 dB in the stopband and from -4.0 dB to -2.5 dB in the passband. Additionally, we demonstrate the robustness of this approach as the ink dyed PDMS filters do not exhibit temporal ageing due to diffusion or autofluorescence. We also show that such filters can easily be integrated in fluorescence systems, with stopbands efficient enough to allow fluorescence measurements under non-optimal conditions (broadband excitation, 180 degrees configuration). Integrated ink dyed PDMS filters add robust optical functionalities to disposable microdevices at a low cost and will enable the use of these devices for a wide range of fluorescence and absorbance based biological and chemical analysis.

Keyword
HOLLOW PRISMS, POLY(DIMETHYLSILOXANE), ABSORPTION, SENSORS, ARRAYS, SYSTEM, CHIP
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-29482 (URN)10.1039/c003575k (DOI)000279851200015 ()2-s2.0-77954619141 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, (FP7/2007-2013)/ERC 209243
Note
QC 20110204Available from: 2011-02-04 Created: 2011-02-02 Last updated: 2017-12-11Bibliographically approved

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