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Sensitive Detection of Bacterial DNA by Magnetic Nanoparticles
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, 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.
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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.

Place, publisher, year, edition, pages
2010. Vol. 82, no 22, 9138-9140 p.
National Category
Analytical Chemistry Other Materials Engineering
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
URN: urn:nbn:se:uu:diva-133693DOI: 10.1021/ac102133eISI: 000284080500002OAI: oai:DiVA.org:uu-133693DiVA: diva2:370069
Available from: 2010-11-15 Created: 2010-11-15 Last updated: 2017-12-12Bibliographically 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. Development of Electrical Readouts for Amplified Single Molecule Detection
Open this publication in new window or tab >>Development of Electrical Readouts for Amplified Single Molecule Detection
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology. 

This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1091
Keyword
Rolling circle amplification, metallization, electrical DNA detection, magnetic nanoparticles, dielectrophoresis, boranephosphonate modified nucleotides, DNA elongation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-247945 (URN)978-91-554-9219-9 (ISBN)
Public defence
2015-05-22, B41, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2015-04-28 Created: 2015-03-25 Last updated: 2015-07-07

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Zardán Gómez de la Torre, TeresaStrömberg, MattiasRussell, CamillaSvedlindh, PeterStrømme, MariaNilsson, Mats

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