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Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
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2012 (English)In: Small, ISSN 1613-6810, Vol. 8, no 14, 2174-2177 p.Article in journal (Refereed) Published
Abstract [en]

A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathogenic simulant of Bacillus anthracis, with a limit-of-detection of 50 spores with a reaction time of 135 min. The study shows the versatility of magnetic nanobeads for detection of biological molecules other than DNA.

Place, publisher, year, edition, pages
2012. Vol. 8, no 14, 2174-2177 p.
Keyword [en]
biosensors, immunoassays, magnetic particles, spores, nanobeads
National Category
Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials; Engineering Science with specialization in Solid State Physics
Identifiers
URN: urn:nbn:se:uu:diva-169224DOI: 10.1002/smll.201102632ISI: 000306362700006PubMedID: 22514097OAI: oai:DiVA.org:uu-169224DiVA: diva2:505650
Available from: 2012-02-24 Created: 2012-02-24 Last updated: 2016-11-30Bibliographically approved
In thesis
1. 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
2. Detection and Sequencing of Amplified Single Molecules
Open this publication in new window or tab >>Detection and Sequencing of Amplified Single Molecules
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes.

In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them.

Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 829
Keyword
padlock probes, proximity ligation assay, rolling circle amplification, circle-to-circle amplification, single molecule detection, amplified single molecules, sequencing, in situ, single cell
National Category
Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-183141 (URN)978-91-554-8508-5 (ISBN)
Public defence
2012-12-06, Rudbecksalen, Dag Hammarskjölds v 20, Rudbecklaboratoriet, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-11-15 Created: 2012-10-23 Last updated: 2013-01-23Bibliographically approved

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Zardán Gómez de la Torre, TeresaKe, RongqinMezger, AnjaSvedlindh, PeterStrømme, MariaNilsson, Mats

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