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Novel diagnostic microarray assay formats towards comprehensive on-site analysis
KTH, School of Biotechnology (BIO), Nano Biotechnology.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , p. xii, 90
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:18
Keywords [en]
magnetic microbeads, gold nanobeads, microarrays, lateral flow, on-site diagnosis, SNPs, pestivirus, autoimmunity, contagious bovine pleuropneumonia, comsol multiphysics, finite element method
National Category
Microbiology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-11221ISBN: 978-91-7415-416-0 (print)OAI: oai:DiVA.org:kth-11221DiVA, id: diva2:242252
Public defence
2009-10-23, FR4 Oskar Klein, AlbaNova Universitetscentrum, Roslagstullsbacken, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-11-23Bibliographically approved
List of papers
1. Visual DNA: Identification of DNA sequence variations by bead trapping
Open this publication in new window or tab >>Visual DNA: Identification of DNA sequence variations by bead trapping
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2007 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 90, p. 741-745Article in journal (Refereed) Published
Abstract [en]

In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.

Keywords
imaging techniques; medical imaging; magnetics; microspheres; oligonucleotide array; sequence analysis; signal amplification assay
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11218 (URN)10.1016/j.ygeno.2007.07.014 (DOI)000251707500010 ()
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Magnetic bead-based detection of autoimmune responses using protein microarrays.
Open this publication in new window or tab >>Magnetic bead-based detection of autoimmune responses using protein microarrays.
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2009 (English)In: New biotechnology, ISSN 1871-6784, Vol. 26, p. 269-276Article in journal (Refereed) Published
Abstract [en]

In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.

Keywords
TECHNOLOGY; DIAGNOSTICS; DISEASES; ASSAY
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11220 (URN)10.1016/j.nbt.2009.07.011 (DOI)000273519000003 ()19664732 (PubMedID)2-s2.0-77952310841 (Scopus ID)
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-01-20Bibliographically approved
3. Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
Open this publication in new window or tab >>Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
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2009 (English)In: Journal of Virological Methods, ISSN 0166-0934, Vol. 155, p. 1-9Article in journal (Refereed) Published
Abstract [en]

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

Keywords
Pestivirus; CSFV; BDV; BVDV1; BVDV2; PCR; Magnetic bead; Microarray
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11219 (URN)10.1016/j.jviromet.2008.04.010 (DOI)000262573400001 ()2-s2.0-57549108180 (Scopus ID)
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-01-20Bibliographically approved
4. A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
Open this publication in new window or tab >>A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
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2010 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 82, no 1, p. 11-18Article in journal (Refereed) Published
Abstract [en]

Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.

Keywords
Lateral flow, Microarray, Gold nanobeads, Contagious bovine pleuropneumonia, Serum analysis, Multiplex, Antigens
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14099 (URN)10.1016/j.mimet.2010.03.007 (DOI)000279567700002 ()2-s2.0-77953541930 (Scopus ID)
Note
QC 20100713Available from: 2010-07-13 Created: 2010-07-13 Last updated: 2017-12-12Bibliographically approved
5. Evaluation of a lateral flow microarray assay system
Open this publication in new window or tab >>Evaluation of a lateral flow microarray assay system
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(English)Article in journal (Other academic) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14101 (URN)
Note
QS 20120326Available from: 2010-07-13 Created: 2010-07-13 Last updated: 2012-03-26Bibliographically approved

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