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Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-4313-1601
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2012 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 7, 3311-3317 p.Article in journal (Refereed) Published
Abstract [en]

The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.

Place, publisher, year, edition, pages
2012. Vol. 84, no 7, 3311-3317 p.
Keyword [en]
Bioactive Paper, Visual Dna, Cellulose, Xyloglucan, Biosensors, Aptamers, Assay
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-95103DOI: 10.1021/ac300025vISI: 000302829800039PubMedID: 22369042Scopus ID: 2-s2.0-84859392980OAI: oai:DiVA.org:kth-95103DiVA: diva2:527415
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20150629

Available from: 2012-05-21 Created: 2012-05-14 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Advances in DNA Detection on Paper Chips
Open this publication in new window or tab >>Advances in DNA Detection on Paper Chips
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice.

The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner).

The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers.

In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. 31 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:19
Keyword
DNA detection, active filter papers, visualization, throughput, fluorescence, superparamagnetic beads
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-133614 (URN)978-91-7501-922-2 (ISBN)
Presentation
2013-11-29, Alfa 4, Kilsbergen, Science for Life Laboratory, Tomtebodavägen 23A, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20131111

Available from: 2013-11-11 Created: 2013-11-07 Last updated: 2013-11-11Bibliographically approved
2. Advances in DNA Detection
Open this publication in new window or tab >>Advances in DNA Detection
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice.

The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work.

The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model, which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. ix, 56 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:4
Keyword
DNA detection, active filter paper, visual detection, throughput, fluorescence, superparamagnetic beads, rare DNA, Q-PCR, chemical fragmentation, DNA library preparation, massively parallel sequencing
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-143047 (URN)978-91-7595-053-2 (ISBN)
Public defence
2014-04-11, CMB Lecture hall, Berzelius väg 21, Karolinska Institute, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140318

Available from: 2014-03-18 Created: 2014-03-16 Last updated: 2014-03-18Bibliographically approved

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