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Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
KTH, School of Biotechnology (BIO). Science for Life Laboratory, SciLifeLab. (Division of Proteomics and Nanobiotechnology)ORCID iD: 0000-0001-7510-0864
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis.

In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics.

Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research.

 

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 69
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:15
Keyword [en]
Droplet microfluidics, Reverse transcription, Droplet PCR, High Throughput biology, Single cell Analysis, Exosomes
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-216669ISBN: 978-91-7729-577-8 (print)OAI: oai:DiVA.org:kth-216669DiVA, id: diva2:1151596
Public defence
2017-11-17, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20171024

Available from: 2017-10-24 Created: 2017-10-23 Last updated: 2017-10-26Bibliographically approved
List of papers
1. Integration of a Droplet-Based Microfluidic System and Silicon Nanoribbon FET Sensor
Open this publication in new window or tab >>Integration of a Droplet-Based Microfluidic System and Silicon Nanoribbon FET Sensor
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2016 (English)In: Micromachines, ISSN 2072-666X, E-ISSN 2072-666X, Vol. 7, no 8Article in journal (Refereed) Published
Abstract [en]

We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.

Place, publisher, year, edition, pages
MDPI AG, 2016
Keyword
NanoFET; silicon nanoribbon; droplet microfluidics; pH measurement
National Category
Nano Technology
Research subject
Information and Communication Technology
Identifiers
urn:nbn:se:kth:diva-191177 (URN)10.3390/mi7080134 (DOI)000382467700006 ()2-s2.0-84984791952 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20160825

Available from: 2016-08-25 Created: 2016-08-25 Last updated: 2017-11-28Bibliographically approved
2. Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
Open this publication in new window or tab >>Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
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2017 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 139, p. 22-28Article in journal (Refereed) Published
Abstract [en]

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Place, publisher, year, edition, pages
Elsevier, 2017
Keyword
Droplet microfluidics, Droplet PCR, Fluorescent beads, Biomarker detection, DNA hybridization
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-211322 (URN)10.1016/j.mimet.2017.04.007 (DOI)000405154900005 ()28434824 (PubMedID)2-s2.0-85018453446 (Scopus ID)
Funder
Swedish Research Council Formas, 221-2011-1692Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170801

Available from: 2017-08-01 Created: 2017-08-01 Last updated: 2017-11-29Bibliographically approved
3. Parallel cDNA synthesis from thousands of individually encapsulated cancer cells: Towards large scale single cell gene expression analysis
Open this publication in new window or tab >>Parallel cDNA synthesis from thousands of individually encapsulated cancer cells: Towards large scale single cell gene expression analysis
2013 (English)In: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, p. 1737-1739Conference paper, Published paper (Refereed)
Abstract [en]

We present microfluidic droplet-based cDNA synthesis of 65 000 individually isolated lung cancer cells in parallel. Cells are encapsulated, individually lysed and the RNA from each cell is reverse transcribed in droplets at a massively parallel scale resulting in thousands of droplets each containing the cells gene expression profile encoded in stable DNA for downstream analysis (figure 1). This could be used for distinguishing between different cell types and study heterogeneity within a cell sample at high throughput scale.

Keyword
CDNA synthesis, Droplet microfluidics, High throughput, Single cell analysis
National Category
Other Biological Topics
Identifiers
urn:nbn:se:kth:diva-168809 (URN)2-s2.0-84907352100 (Scopus ID)
Conference
17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany
Note

QC 20150610

Available from: 2015-06-10 Created: 2015-06-09 Last updated: 2017-10-24Bibliographically approved
4. Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content
Open this publication in new window or tab >>Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a workflow for reverse transcription-PCR (RT-PCR) in microfluidic droplets to identify exosomes based on their RNA content. Available techniques for exosome detection have been limited to size or surface markers which limit their diagnostic capabilities. Exosome detection based on RNA content could be developed to be used as a diagnostic, prognostic or predictive tool for cancer based on specific RNA biomarkers in liquid biopsies. In this manuscript we demonstrate a high throughput method for the amplification of exosome derived 18S RNA in microfluidic droplets. Automated image analysis using open source software was applied to distinguish and count PCR-positive droplets with fluorescent intensity over a set threshold. We benchmark our workflow against picoliter scale RT-PCR on serially diluted exosome samples and demonstrate the ability of the droplet based workflow to correctly rank exosome samples based on exosome concentration.  This represents a key step towards a quantitative analysis of exosomal RNA content and the sorting of single exosomes by their RNA content.

Keyword
Droplet Microfluidics, Exosomes, RT-PCR
National Category
Diagnostic Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-216586 (URN)
Note

QC 20171023

Available from: 2017-10-23 Created: 2017-10-23 Last updated: 2017-10-24Bibliographically approved

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