Dufva, Martin

Guldberg, Per

Hansen, Mikkel Fougt

Lee, Jung-Rok

Rizzi, Giovanni

Wang, Shan X.

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 18, 2017Denaturing strategies for detection of double stranded PCR products on GMRmagnetic sensorsRizzi, Giovanni; Lee, Jung-Rok; Guldberg, Per; Dufva, Martin; Wang, Shan X.; Hansen, Mikkel FougtPublication date:2016Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Rizzi, G., Lee, J-R., Guldberg, P., Dufva, M., Wang, S. X., & Hansen, M. F. (2016). Denaturing strategies fordetection of double stranded PCR products on GMR magnetic sensors. Abstract from Biosensors 2016,Gothenburg, Sweden.Important notes:  Do NOT write outside the grey boxes. Any text or images outside the boxes will be deleted.  Do NOT alter the structure of this form. Simply enter your information into the boxes. The form will be automatically processed – if you alter its structure your submission will not be processed correctly.  Do not include keywords – you can add them when you submit the abstract online.   Title: Denaturing strategies for detection of double stranded PCR products on GMR magnetic sensors  Authors & affiliations: Giovanni Rizzi1, Jung-Rok Lee2, Per Guldberg3,Martin Dufva1, Shan X. Wang2,Mikkel F. Hansen1 1. DTU Nanotech, Technical University of Denmark, Kgs. Lyngby, Denmark. 2. Geballe Laboratory for Advanced Materials, Stanford University, Stanford CA. 3. The Danish Cancer Society, Copenhagen, Denmark.  Abstract:  (Your abstract must use Normal style and must fit in this box.  Your abstract should be no longer than 300 words. The box will ‘expand’ over 2 pages as you add text/diagrams into it.) Preparation of Your Abstract 1. The title should be as brief as possible but long enough to indicate clearly the nature of the study. Capitalise the first letter of the first word ONLY (place names excluded). No full stop at the end. 2. Abstracts should state briefly and clearly the purpose, methods, results and conclusions of the work. Introduction: Clearly state the purpose of the abstract Methods: Describe your selection of observations or experimental subjects clearly Results: Present your results in a logical sequence in text, tables and illustrations Discussion: Emphasize new and important aspects of the study and conclusions that are drawn from them Arrays of GMR magnetic field sensors have been demonstrated for the detection of proteins1 and DNA2. The readout is based on the detection of the target-mediated binding of magnetic nanoparticle (MNP) labels to the sensor surface. The assay is insensitive to the sample matrix as there is virtually no detectable magnetic response from biological samples.   Here, we employ the GMR array platform to detect PCR products from melanoma cell lines, with the final goal of profiling mutations of diagnostic relevance3. The sensor surface is functionalized with ssDNA probes. The forward PCR primers are biotinylated to facilitate binding to streptavidin coated MNPs. The dsDNA product has to be denatured to enable target binding to the sensor surface. We aim to obtain the highest binding signal, as specificity can be increased by optimizing the stringency condition during washing4.  In this work, we tested two approaches for the denaturation of PCR products and magnetic labelling:  (1) heat denaturation followed by shock cooling, on-chip hybridization and on-chip labelling with MNPs (Fig.1a).  (2) labelling of dsDNA PCR products with MNPs, immobilization of MNPs in a magnetic separation column, denaturation in 6M urea in DI water at 75°C, release of MNPs with ssDNA labels, on-chip detection (Fig.1b).   Figure 1c and Fig.1d show the GMR signal from experiments using heat denaturation and magnetic column separation, respectively. In Fig.1c the binding is faster because we measure the biotin-streptavidin binding, whereas the binding in Fig.1d is limited by the DNA hybridisation and diffusion of beads with targets. Both methods offer high signals with small deviations. The denaturation in magnetic column results in slightly higher signal due to the complete removal of reverse complement. Both techniques are viable for detection of PCR products on GMR sensors, the choice will be driven by a trade-off between assay time and signal intensity.   References: 1-Gaster, Richard S., et al., 2009, Nature medicine 15(11) 1327-1332. 2-Xu, Liang, et al., 2008, Biosensors and Bioelectronics 24(1) 99-103. 3-Dahl, Christina, et al., 2013, Molecular Cancer Research 11(10) 1166-1178. 4-Rizzi, Giovanni, et al., 2015, Journal of Magnetism and Magnetic Materials 380 215-220. Important notes:  Do NOT write outside the grey boxes. Any text or images outside the boxes will be deleted.  Do NOT alter the structure of this form. Simply enter your information into the boxes. The form will be automatically processed – if you alter its structure your submission will not be processed correctly.  Do not include keywords – you can add them when you submit the abstract online.    Figure 1: Two approaches to obtain ssDNA from double stranded PCR products for detection on GMR sensors. The forward primers are biotinylated to bind streptavidin-coated magnetic nanoparticles (MNPs). (a) Heat denaturation. The sample is heated to 90°C and cooled abruptly to inhibit re-hybridisation. The sample is then incubated on the sensor surface at 37°C for 60 min. After washing, the chip is mounted in the system and the signal is monitored during labelling with MNPs. (b) Separation in magnetic column. The sample is mixed with MNPs for labelling (30min) and then immobilized in a magnetic column. The reverse strand is denatured with 6M urea in DI buffer at 75°C. The labelled forward strand is re-suspended in 2xSSC buffer and injected over the sensors. The hybridization is monitored in real-time. (c) Signal measured during labelling after heat denaturation. (d) Signal measured during hybridization after separation in magnetic column. Four sensors were functionalized with each probe.     

Denaturing strategies for detection of double stranded PCR products on GMR magnetic sensors

http://orbit.dtu.dk/ws/files/127591149/Abstract_v2_1.pdf

Denaturing strategies for detection of double stranded PCR products on GMR magnetic sensors

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology