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Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
2015 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 67, no SI, 11-17 p.Article in journal (Refereed) Published
Abstract [en]

Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.

Place, publisher, year, edition, pages
2015. Vol. 67, no SI, 11-17 p.
Keyword [en]
Padlock probe, RCA, Single molecule detection, Resistive pulse sensing, Nanopore
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-248795DOI: 10.1016/j.bios.2014.06.040ISI: 000350076900003PubMedID: 25000851OAI: oai:DiVA.org:uu-248795DiVA: diva2:801885
Available from: 2015-04-10 Created: 2015-04-08 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Single Molecule Detection: Microfluidic Automation and Digital Quantification
Open this publication in new window or tab >>Single Molecule Detection: Microfluidic Automation and Digital Quantification
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity.

This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1189
Keyword
single molecule, digital, rolling circle amplification, magnetic particle, padlock probe, microfluidics, resistive pulse sensing, lab on chip, mobile phone microscopy, enrichment, sequencing
National Category
Cell and Molecular Biology Medical Genetics Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:uu:diva-279372 (URN)978-91-554-9498-8 (ISBN)
Public defence
2016-04-22, B41, BMC, Husargatan 3, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2016-03-31 Created: 2016-03-01 Last updated: 2016-04-04

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Kuhnemund, MalteNilsson, Mats

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