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Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0003-4313-1601
Vise andre og tillknytning
2012 (engelsk)Inngår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, nr 7, s. 3311-3317Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2012. Vol. 84, nr 7, s. 3311-3317
Emneord [en]
Bioactive Paper, Visual Dna, Cellulose, Xyloglucan, Biosensors, Aptamers, Assay
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-95103DOI: 10.1021/ac300025vISI: 000302829800039PubMedID: 22369042Scopus ID: 2-s2.0-84859392980OAI: oai:DiVA.org:kth-95103DiVA, id: diva2:527415
Forskningsfinansiär
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20150629

Tilgjengelig fra: 2012-05-21 Laget: 2012-05-14 Sist oppdatert: 2017-12-07bibliografisk kontrollert
Inngår i avhandling
1. Advances in DNA Detection on Paper Chips
Åpne denne publikasjonen i ny fane eller vindu >>Advances in DNA Detection on Paper Chips
2013 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2013. s. 31
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2013:19
Emneord
DNA detection, active filter papers, visualization, throughput, fluorescence, superparamagnetic beads
HSV kategori
Forskningsprogram
SRA - Molekylär biovetenskap
Identifikatorer
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 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20131111

Tilgjengelig fra: 2013-11-11 Laget: 2013-11-07 Sist oppdatert: 2013-11-11bibliografisk kontrollert
2. Advances in DNA Detection
Åpne denne publikasjonen i ny fane eller vindu >>Advances in DNA Detection
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2014. s. ix, 56
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:4
Emneord
DNA detection, active filter paper, visual detection, throughput, fluorescence, superparamagnetic beads, rare DNA, Q-PCR, chemical fragmentation, DNA library preparation, massively parallel sequencing
HSV kategori
Forskningsprogram
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-143047 (URN)978-91-7595-053-2 (ISBN)
Disputas
2014-04-11, CMB Lecture hall, Berzelius väg 21, Karolinska Institute, Solna, 10:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20140318

Tilgjengelig fra: 2014-03-18 Laget: 2014-03-16 Sist oppdatert: 2014-03-18bibliografisk kontrollert

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