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Interbead interactions within oligonucleotide functionalized ferrofluids suitable for magnetic biosensor applications
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2007 (English)In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 40, no 5, 1320-1330 p.Article in journal (Refereed) Published
Abstract [en]

Interactions in oligonucleotide functionalized ferrofluids were investigated by measurements of frequency dependent complex magnetization in a superconducting quantum interference device. The ferrofluids consisted of aqueous suspensions of single-stranded oligonucleotide functionalized 130 nm sized magnetic beads, suitable for further use in magnetic biosensor applications based on changes in the Brownian relaxation behaviour of oligonucleotide-coated beads. The interbead interactions were found to depend strongly on the surface coverage by oligonucleotide molecules. At low surface coverage, aggregates of beads within the ferrofluid were formed, most likely due to interbead SS-crosslinking reactions. When the surface coverage increased, the entropic interbead repulsion arising from the thermal motion of oligonucleotide molecules began to stabilize the ferrofluid by preventing aggregation, and the crosslinking probability decreased. At a surface coverage, of ~40 oligonucleotides per bead, determined by a radioactive labelling analysis, an optimal configuration was obtained for which the ferrofluid behaved as consisting of almost non-interacting beads. At higher oligonucleotide functionalization degrees, the high coverage induced less thermal motion flexibility of the oligonucleotide chains which decreased the entropic repulsion effect. This in turn led to a higher crosslinking probability, as well as an increase in the hydrodynamic size of the beads. Thus, the ferrofluid sample of nearly non-interacting nature, in which an optimal degree of stabilization and the highest Brownian relaxation frequency are achieved in the low surface coverage region, may be considered as the most appropriate one for further use in magnetic biosensor applications, from both a functional and an economic point of view.

Place, publisher, year, edition, pages
2007. Vol. 40, no 5, 1320-1330 p.
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
URN: urn:nbn:se:uu:diva-98019DOI: 10.1088/0022-3727/40/5/002ISI: 000245283100022OAI: oai:DiVA.org:uu-98019DiVA: diva2:173176
Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Molecular Diagnostics Using Volume-Amplified Magnetic Nanobeads: Towards the Development of a Novel Biosensor System
Open this publication in new window or tab >>Molecular Diagnostics Using Volume-Amplified Magnetic Nanobeads: Towards the Development of a Novel Biosensor System
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Micro- or nanometer sized magnetic particles (beads) currently have a vast range of life science applications in, for example, bioseparation techniques, cancer therapy, development of contrast agents and biosensing techniques. In the latter field, magnetic beads offer several unique advantages, including minimal background signals, physical and chemical stability and low manufacturing costs. Because of these properties, magnetic biosensing techniques are potential candidates for low-cost, easy-to-use molecular diagnostic devices.

This doctoral thesis focuses mainly on the proof of principle and further development of a new magnetic biosensor platform for detection of DNA targets, a potential candidate for a new generation of low-cost, easy-to-use diagnostic devices: the Volume-Amplified Magnetic Nanobead Detection Assay (VAM-NDA). The VAM-NDA principle combines target recognition by padlock probe ligation followed by rolling circle amplification (RCA) of the reacted probes with changes in Brownian relaxation behaviour of magnetic nanobeads (typically ~100 nm in diameter) induced by a change in hydrodynamic bead volume. More specifically, the RCA products (coils, typically ~1 μm in diameter) are detected magnetically by adding magnetic beads tagged with detection probes complementary to part of the repeating RCA-coil sequence. Thus, depending on the target concentration, a certain quantity of beads binds to the coils by base-pair hybridisation (bead immobilisation), resulting in a dramatic bead volume increase, which is then detected by measuring the complex magnetisation spectrum. Use of a commercial SQUID magnetometer for measuring complex magnetisation resulted in a detection limit in the low pM range for DNA targets with excellent quantification accuracy. Simultaneous multiplexing was also evaluated.

The stability and aging of typical commercial ferrofluids (suspensions of magnetic beads) were investigated by measuring the complex magnetisation of and interbead interactions in oligonucleotide-functionalised ferrofluids. In summary, the bead surface characteristics were found to have a strong impact on the measured dynamic magnetic properties.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2009. 102 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 598
Keyword
Magnetic Biosensors, Ferrofluid, Biofunctionalisation, Ferrofluidic Aging, Interbead Interactions, Magnetic Nanobeads, Complex Magnetisation, Brownian Relaxation, Padlock Probes, Rolling Circle Amplification
National Category
Other Engineering and Technologies
Identifiers
urn:nbn:se:uu:diva-9542 (URN)978-91-554-7402-7 (ISBN)
Public defence
2009-03-06, Polhemsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2009-06-22Bibliographically approved
2. Microfluidic and Molecular Tools for Genetic Analyses
Open this publication in new window or tab >>Microfluidic and Molecular Tools for Genetic Analyses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments.

The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences.

A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping.

The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 36 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 546
Keyword
microfluidics, PCR, sequencing, cancer genetics
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-121536 (URN)978-91-554-7765-3 (ISBN)
Public defence
2010-05-05, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-14 Created: 2010-03-24 Last updated: 2018-01-12Bibliographically approved

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Nanotechnology and Functional MaterialsSolid State PhysicsDepartment of Genetics and Pathology
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