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Sensitive molecular diagnostics using volume-amplified magnetic nanobeads
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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.
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2008 (English)In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 8, no 3, 816-821 p.Article in journal (Refereed) Published
Abstract [en]

In this letter, we demonstrate a new principle for diagnostics based on DNA sequence detection using single-stranded oligonucleotide tagged magnetic nanobeads. The target DNA is recognized and volume-amplified to large coils by circularization of linear padlock probes through probe hybridization and ligation, followed by rolling circle amplification (RCA). Upon hybridization of the nanobeads in the RCA coils, the complex magnetization spectrum of the beads changes dramatically, induced by the attached volume-amplified target molecules. We show that the magnetization spectrum of the nanobeads can be used for concentration determination of RCA coils down to the pM range, thus creating the opportunity for nonfluorescence-based cost-efficient high-sensitivity diagnostics tool. We also show that the bead incorporation in the coils is diffusion-controlled and consequently may be accelerated by incubating the sample at higher temperatures.

Place, publisher, year, edition, pages
2008. Vol. 8, no 3, 816-821 p.
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Solid State Physics; Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
URN: urn:nbn:se:uu:diva-98020DOI: 10.1021/nl072760eISI: 000253947400009PubMedID: 18247520OAI: oai:DiVA.org:uu-98020DiVA: diva2:173177
Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2016-11-30Bibliographically approved
In thesis
1. Readout Strategies for Biomolecular Analyses
Open this publication in new window or tab >>Readout Strategies for Biomolecular Analyses
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 41 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 563
Keyword
Padlock probe, Selector probe, Rolling circle amplification, Single molecule detection, Amplified single molecule, Random array
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-9343 (URN)978-91-554-7320-4 (ISBN)
Public defence
2008-11-27, Rudbecksalen, Rudbeck Laboratory, C11, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2008-11-04 Created: 2008-11-04 Last updated: 2009-07-07Bibliographically approved
2. 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

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Strömberg, MattiasGunnarsson, KlasSvedlindh, PeterStrømme, Maria

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