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Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
2014 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 16, 2983-2992 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.

Place, publisher, year, edition, pages
2014. Vol. 14, no 16, 2983-2992 p.
National Category
Medical Biotechnology
URN: urn:nbn:se:uu:diva-231103DOI: 10.1039/c4lc00348aISI: 000339470400013PubMedID: 24934991OAI: oai:DiVA.org:uu-231103DiVA: diva2:744185
Available from: 2014-09-07 Created: 2014-09-04 Last updated: 2016-04-04Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1189
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
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)
Available from: 2016-03-31 Created: 2016-03-01 Last updated: 2016-04-04

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