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Detection of Biomolecules Using Volume-Amplified Magnetic Nanobeads
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
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 [en]
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: urn:nbn:se:uu:diva-169431ISBN: 978-91-554-8288-6 (print)OAI: oai:DiVA.org:uu-169431DiVA: diva2:506616
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
List of papers
1. Microscopic mechanisms influencing the volume amplified magnetic nanobead detection assay
Open this publication in new window or tab >>Microscopic mechanisms influencing the volume amplified magnetic nanobead detection assay
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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.

Keyword
Bioassay development, Brownian relaxation, Padlock probes, Rolling circle amplification, Probe-tagged magnetic beads, Microscopic mechanisms
National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-98021 (URN)10.1016/j.bios.2008.06.043 (DOI)000261262000030 ()18703330 (PubMedID)
Available from: 2009-02-13 Created: 2009-02-13 Last updated: 2017-12-14Bibliographically approved
2. Multiplex Detection of DNA Sequences Using the Volume-Amplified Magnetic Nanobead Detection Assay
Open this publication in new window or tab >>Multiplex Detection of DNA Sequences Using the Volume-Amplified Magnetic Nanobead Detection Assay
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2009 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 9, 3398-3406 p.Article in journal (Refereed) Published
Abstract [en]

The possibility for conducting multiplex detection of DNA-sequences using the volume-amplified magnetic nanobead detection assay[Stromberg, M.; Goransson, J.; Gunnarsson, K; Nilsson, M.; Svedlindh, P.; Stromme, M. Nano Lett. 2008, 8, 816-821] was investigated. In this methodology, a batch consisting of a mixture of several sizes of probe-tagged magnetic beads was used for detection of several types of  targets in the same compartment Furthermore, a nonlinear least-squares deconvolution procedure of the composite imaginary part of complex   magnetization vs frequency spectra based on the Cole-Cole model was   applied to analyze the data. The results of a quantitative biplex analysis experiment were compared with the corresponding separate   single-target assays. Finally, triplex analysis was briefly demonstrated qualitatively. Biplex and triplex detection were found to perform well qualitatively. Biplex detection was found to enable a rough target quantification. Multiplex detection may become a  complement to performing multiple separate single-target assays for, e.g., parallel detection of multiple infectious pathogens. Multiplex detection also permits robust relative quantification and inclusion of an internal control to improve quantification accuracy.

National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-102082 (URN)10.1021/ac900561r (DOI)000265632400029 ()19334737 (PubMedID)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13Bibliographically approved
3. Investigation of Immobilization of Functionalized Magnetic Nanobeads in Rolling CircleAmplified DNA Coils
Open this publication in new window or tab >>Investigation of Immobilization of Functionalized Magnetic Nanobeads in Rolling CircleAmplified DNA Coils
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2010 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 10, 3707-3713 p.Article in journal (Refereed) Published
Abstract [en]

Immobilization characteristics for single-stranded oligonucleotide- functionalized magnetic beads with nominal sizes of 40, 80, 130, and 250 nm in rolling circle amplified (RCA) DNA coils is investigated by employing complex magnetization measurements, dynamic light scattering and fluorescence microscopy. It was found that larger beads in a polydisperse bead size distribution more easily immobilize in the RCA DNA coils than do smaller beads. This may be related to a higher oligonucleotide surface coverage for the larger beads. Furthermore, it was concluded that both bead size and oligonucleotide surface coverage determine whether beads immobilize to give isolated coils with beads or larger clusters of beads and coils. A small bead size and a low oligonucleotide surface coverage favor the first kind of immobilization behavior, whereas a large bead size and a high oligonucleotide surface coverage favor the other. The present findings could be used to optimize both size and surface functionalization of beads employed in substrate-free magnetic biosensors.

National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-120765 (URN)10.1021/jp911251k (DOI)000275328400037 ()20175549 (PubMedID)
Available from: 2010-03-16 Created: 2010-03-16 Last updated: 2017-12-12Bibliographically approved
4. Sensitive Detection of Bacterial DNA by Magnetic Nanoparticles
Open this publication in new window or tab >>Sensitive Detection of Bacterial DNA by Magnetic Nanoparticles
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2010 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 22, 9138-9140 p.Article in journal, Letter (Refereed) Published
Abstract [en]

This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.

National Category
Analytical Chemistry Other Materials Engineering
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-133693 (URN)10.1021/ac102133e (DOI)000284080500002 ()
Available from: 2010-11-15 Created: 2010-11-15 Last updated: 2017-12-12Bibliographically approved
5. Evaluation of the Sulfo-Succinimidyl-4-(N-Maleimidomethyl) Cyclohexane-1-Carboxylate coupling chemistry for attachment of oligonucleotides to magnetic nanobeads
Open this publication in new window or tab >>Evaluation of the Sulfo-Succinimidyl-4-(N-Maleimidomethyl) Cyclohexane-1-Carboxylate coupling chemistry for attachment of oligonucleotides to magnetic nanobeads
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2011 (English)In: Journal of Nanoscience and Nanotechnology, ISSN 1533-4880, E-ISSN 1533-4899, Vol. 11, no 10, 8532-8537 p.Article in journal (Refereed) Published
Abstract [en]

The sulfo-SMCC (Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) coupling chemistry was evaluated for immobilization of oligonucleotides onto 130 nm sized magnetic nanobeads aimed for bio-detection in a magnetic readout assay. The chemistry was found to produce a high surface coverage of similar to 93 +/- 10 oligonucleotides per bead whereas stability tests showed that about 50% of the oligonucleotides detached from the bead surfaces after eight weeks of storage in a buffer solution. It was shown that bead aggregation prior to magnetic readout could be suppressed by incubating the samples at 70 degrees C for 30 min. The same temperature was also shown to be the most favorable for hybridization between the oligonucleotide functionalized beads and rolling circle amplified DNA molecules. This should simplify the heating procedure in a biosensor in which hybridization and magnetic readout is performed in the same compartment.

Place, publisher, year, edition, pages
American Scientific Publishers, 2011
Keyword
DNA Detection, Oligonucleotide Coupling Chemistry, Sulfo-SMCC, Magnetic Nanobeads, Biosensor
National Category
Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-161323 (URN)10.1166/jnn.2011.5667 (DOI)000298363900014 ()
Available from: 2011-11-11 Created: 2011-11-11 Last updated: 2017-12-08Bibliographically approved
6. Detection of rolling circle amplified DNA molecules using probe-tagged magnetic nanobeads in a portable AC susceptometer
Open this publication in new window or tab >>Detection of rolling circle amplified DNA molecules using probe-tagged magnetic nanobeads in a portable AC susceptometer
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2011 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 29, no 1, 195-199 p.Article in journal (Refereed) Published
Abstract [en]

Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250 nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID).

It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ∼4 pM for all bead sizes under study.

The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.

Keyword
Probe-tagged magnetic beads; Padlock probes; Rolling circle amplification; Brownian relaxation; AC susceptometer
National Category
Nano Technology Engineering and Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials; Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-159175 (URN)10.1016/j.bios.2011.08.019 (DOI)000296174700031 ()
Available from: 2011-09-23 Created: 2011-09-23 Last updated: 2017-12-08Bibliographically approved
7. Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
Open this publication in new window or tab >>Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
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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.

Keyword
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:nbn:se:uu:diva-169224 (URN)10.1002/smll.201102632 (DOI)000306362700006 ()22514097 (PubMedID)
Available from: 2012-02-24 Created: 2012-02-24 Last updated: 2016-11-30Bibliographically approved

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  • modern-language-association-8th-edition
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  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
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  • Other locale
More languages
Output format
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  • asciidoc
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