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Microscopic mechanisms influencing the volume amplified magnetic nanobead detection assay
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, 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.
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2008 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 24, no 4, 696-703 p.Article in journal (Refereed) Published
Abstract [en]

The volume amplified magnetic nanobead detection assay [Strömberg, M., Göransson, J., Gunnarsson, K., Nilsson, M., Svedlindh, P., Strømme, M., 2008. Nano Letters 8, 816-821] was investigated with respect to bead size, bead surface coverage of probe oligonucleotides, bead concentration and rolling circle amplification (RCA) time, with the objective to improve the understanding of the microscopic mechanisms influencing the assay. The most important findings for future biosensor development were the following: (i) small beads exhibit a much reduced tendency to cross-link several RCA products, thus enabling use of both complex magnetisation turn-on and turn-off detection strategies, whereas larger beads only allow for turn-off detection; (ii) small beads exhibit faster immobilisation kinetics, thus reducing the time for diagnostic test completion, and also immobilise in larger numbers than larger beads. Finally, (iii) by demonstrating qualitative dual-target detection of bacterial DNA sequences using 130 and 250nm beads, the bioassay was shown to allow for multiplexed detection.

Place, publisher, year, edition, pages
2008. Vol. 24, no 4, 696-703 p.
Keyword [en]
Bioassay development, Brownian relaxation, Padlock probes, Rolling circle amplification, Probe-tagged magnetic beads, Microscopic mechanisms
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-98021DOI: 10.1016/j.bios.2008.06.043ISI: 000261262000030PubMedID: 18703330OAI: oai:DiVA.org:uu-98021DiVA: diva2:173178
Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2016-11-30Bibliographically 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. Detection of Biomolecules Using Volume-Amplified Magnetic Nanobeads
Open this publication in new window or tab >>Detection of Biomolecules Using Volume-Amplified Magnetic Nanobeads
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes a new approach to biomolecular analysis, called the volume-amplified magnetic nanobead detection assay (VAM-DNA). It is a sensitive, specific magnetic bioassay that offers a potential platform for the development of low-cost, easy-to-use diagnostic devices. The VAM-NDA consists of three basic steps: biomolecular target recognition, enzymatic amplification of the probe-target complex using the rolling circle amplification (RCA) technique, and addition of target complementary probe-tagged magnetic nanobeads which exhibit Brownian relaxation behavior. Target detection is demonstrated by measuring the frequency-dependent complex magnetization of the magnetic beads. The binding of the RCA products (target DNA-sequence coils) to the bead surface causes a dramatic increase in the bead size, corresponding essentially to the size of the DNA coil (typically around one micrometer). This causes a decrease in the Brownian relaxation frequency, since it is inversely proportional to the hydrodynamic size of the beads. The concentration of the DNA coils is monitored by measuring the decrease in amplitude of the Brownian relaxation peaks of free beads.

The parameters oligonucleotide surface coverage, bead concentration, bead size and RCA times were investigated in this thesis to characterize features of the assay. It was found that all of these parameters affect the outcome and efficiency of the assay.

The possibility of implementing the assay on a portable, highly sensitive AC susceptometer platform was also investigated. The performance of the assay under these circumstances was compared with that using a superconducting quantum interference device (SQUID); the sensitivity of the assay was similar for both platforms. It is concluded that, the VAM-NDA opens up the possibility to perform biomolecular detection in point-of-care and outpatient settings on portable platforms similar to the one tested in this thesis.

Finally, the VAM-NDA was used to detect Escherichia coli bacteria and the spores of Bacillus globigii, the non-pathogenic simulant of Bacillus anthracis. A limit of detection of at least 50 bacteria or spores was achieved. This shows that the assay has great potential for sensitive detection of biomolecules in both environmental and biomedical applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 905
Keyword
Magnetic biosensor, magnetic nanobeads, Brownian relaxation, padlock probe, rolling circle amplification, DNA detection, protein detection
National Category
Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-169431 (URN)978-91-554-8288-6 (ISBN)
Public defence
2012-04-13, Å 2005, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2012-03-23 Created: 2012-02-29 Last updated: 2012-03-29Bibliographically approved

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Strömberg, MattiasZardán Gómez de la Torre, TeresaGunnarsson, KlasStrømme, MariaSvedlindh, Peter

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