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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-146355OAI: oai:DiVA.org:su-146355DiVA: diva2:1136823
Available from: 2017-08-29 Created: 2017-08-29 Last updated: 2017-08-29Bibliographically approved
In thesis
1. Nucleic acid analysis tools: Novel technologies and biomedical applications
Open this publication in new window or tab >>Nucleic acid analysis tools: Novel technologies and biomedical applications
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth.

Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases.

Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections.

These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017
Keyword
Nucleic acid analysis, padlock probes, selector probes, rolling circle amplification, microfluidics, single-molecule detection, digital quantification, fluorescence microscopy, mobile phone microscopy, molecular diagnostics, virology
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-146334 (URN)978-91-7649-905-4 (ISBN)978-91-7649-906-1 (ISBN)
Public defence
2017-10-12, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.

Available from: 2017-09-20 Created: 2017-08-29 Last updated: 2017-09-20Bibliographically approved

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