Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Droplet microfluidics for single cell and nucleic acid analysis
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0003-3618-9944
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening.

The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. , 58 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:17
Keyword [en]
Acoustophoresis, Biomarker detection, Cell behavior analysis, Cell factories, Droplet microfluidics, Droplet PCR, High throughput biology, Label-free enrichment, Single cell analysis
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-192668ISBN: 978-91-7729-125-1 (print)OAI: oai:DiVA.org:kth-192668DiVA: diva2:971827
Public defence
2016-10-21, Gard-aulan, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20160926

Available from: 2016-09-26 Created: 2016-09-19 Last updated: 2016-09-26Bibliographically approved
List of papers
1. Automated analysis of dynamic behavior of single cells in picoliter droplets
Open this publication in new window or tab >>Automated analysis of dynamic behavior of single cells in picoliter droplets
Show others...
2014 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 5, 931-937 p.Article in journal (Refereed) Published
Abstract [en]

We present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells over time. This high-throughput assay allows encapsulation of single cells in micro-droplets and traps intact droplets in arrays of miniature wells on a PDMS-glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allows the determination of the viability of individual cells over time. In order to automatically track the droplets containing cells, we developed a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, we studied the viability of several hundred single isolated HEK293T cells over time and demonstrated a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single cell analysis, e.g. monitoring heterogeneity of drug action over time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro.

Keyword
Microfluidic System, Flow-Cytometry, In-Vitro, Expression, Time, Heterogeneity, Device, Encapsulation, Tracking, Kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-129290 (URN)10.1039/c3lc51136g (DOI)000330784400013 ()2-s2.0-84893475740 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceFormasSwedish Research Council
Note

QC 20140228. Updated from manuscript to article in journal.

Available from: 2013-09-26 Created: 2013-09-25 Last updated: 2017-12-06Bibliographically approved
2. Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation
Open this publication in new window or tab >>Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation
2016 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683Article in journal (Refereed) Epub ahead of print
Abstract [en]

The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2016
National Category
Bioprocess Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-192574 (URN)10.1002/elps.201600316 (DOI)000393298200009 ()2-s2.0-84987648085 (Scopus ID)
Note

QC 20160921

Available from: 2016-09-15 Created: 2016-09-15 Last updated: 2017-11-21Bibliographically approved
3. Color-coded bead based readout from droplet PCR for the detection of pathogen biomarkers
Open this publication in new window or tab >>Color-coded bead based readout from droplet PCR for the detection of pathogen biomarkers
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a workflow using fluorescent color-coded Luminex beads to detect the

outcome of droplet PCR assay. The assay was performed to detect three important

poultry pathogens: avian influenza, infectious laryngotracheitis virus and

campylobacter. Droplet-based TaqMan PCR has been commonly used for detection

of rare and significant biomarkers in clinical samples. However, the spectral overlap

of fluorescent TaqMan probes limits the detection to 5 different targets in a single

assay. The color codes of the Luminex detection beads allowed accurate classification

of the different bead sets used in this assay concurrently. The target-specific capture

probes coupled to distinct bead sets enabled capture and detection of target DNA in

the droplet. The capture assay detected target DNA of all three poultry pathogens with

high specificity, from samples with average target concentration of 1 template per

droplet. This workflow demonstrates that the detection panel of droplet PCR assay

can be increased to potentially detect multiple targets in a sample by utilizing the

scalability offered by the color-coded detection beads.

National Category
Diagnostic Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-192575 (URN)
Note

QC 20160921

Available from: 2016-09-15 Created: 2016-09-15 Last updated: 2016-09-21Bibliographically approved
4. Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis
Open this publication in new window or tab >>Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis
Show others...
2015 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 20, 10521-10526 p.Article in journal (Refereed) Published
Abstract [en]

In this paper, we utilize bulk acoustic waves to control the position of micropartides inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the micropartides. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of micropartides during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated micropartides to the center of the droplets. The method is generic, requires no labeling of the micropartides, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 mu m in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich micropartides and open up the possibility for new droplet-based assays that are not performed today.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2015
Keyword
Bioassay, Biochips, Microarrays, Microfluidics, Position control, Acoustic standing wave, Biochemical assay, Bulk acoustic waves, Daughter droplets, Droplet microfluidics, Micro fluidic system, Polystyrene beads, Two phase systems
National Category
Robotics Other Electrical Engineering, Electronic Engineering, Information Engineering
Identifiers
urn:nbn:se:kth:diva-176960 (URN)10.1021/acs.analchem.5b02746 (DOI)000363348300051 ()26422760 (PubMedID)2-s2.0-84945288371 (Scopus ID)
Note

QC 20151216

Available from: 2015-12-16 Created: 2015-11-13 Last updated: 2017-12-01Bibliographically approved

Open Access in DiVA

fulltext(8300 kB)177 downloads
File information
File name FULLTEXT01.pdfFile size 8300 kBChecksum SHA-512
d51d303cf48c54a02d0ede004a9daa3d736c74b9024c2ff4ca4457ede2e1b302542bea9f846644f22b56e09b1aba8c44353fc1b12f21f5110ce4df4bcd0e38d5
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Periyannan Rajeswari, Prem Kumar
By organisation
Proteomics and Nanobiotechnology
Other Engineering and Technologies not elsewhere specified

Search outside of DiVA

GoogleGoogle Scholar
Total: 177 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1280 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf