title: Denaturation strategies for detection of double stranded PCR products on GMR magnetic biosensor array.
abstract: Microarrays and other surface-based nucleic acid detection schemes rely on the hybridization of the target to surface-bound detection probes. We present the first comparison of two strategies to detect DNA using a giant magnetoresistive (GMR) biosensor platform starting from an initially double-stranded DNA target. The target strand of interest is biotinylated and detected by the GMR sensor by linking streptavidin magnetic nanoparticles (MNPs) to the sensor surface. The sensor platform has a dynamic detection range from 40pM to 40nM with highly reproducible results and is used to monitor real-time binding signals. The first strategy, using off-chip heat denaturation followed by sequential on-chip incubation of the nucleic acids and MNPs, produces a signal that stabilizes quickly but the signal magnitude is reduced due to competitive rehybridization of the target in solution. The second strategy, using magnetic capture of the double-stranded product followed by denaturing, produces a higher signal but the signal increase is limited by diffusion of the MNPs. Our results show that both strategies give highly reproducible results but that the signal obtained using magnetic capture is higher and insensitive to rehybridization.
authors.affiliation: Department of Micro, and Nanotechnology, DTU Nanotech, Technical University of Denmark, Building 345B, DK-2800 Kgs., Lyngby, Denmark. Electronic address: giori@nanotech.dtu.dk.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Rizzi
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford CA, USA. Electronic address: jungrok@stanford.edu.
authors.forename: Jung-Rok
authors.initials: JR
authors.lastname: Lee
authors.affiliation: Diet, Genes and Environment, Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Electronic address: perg@cancer.dk.
authors.forename: Per
authors.initials: P
authors.lastname: Guldberg
authors.affiliation: Department of Micro, and Nanotechnology, DTU Nanotech, Technical University of Denmark, Building 345B, DK-2800 Kgs., Lyngby, Denmark. Electronic address: martin.dufva@nanotech.dtu.dk.
authors.forename: Martin
authors.initials: M
authors.lastname: Dufva
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford CA, USA. Electronic address: sxwang@stanford.edu.
authors.forename: Shan X
authors.initials: SX
authors.lastname: Wang
authors.affiliation: Department of Micro, and Nanotechnology, DTU Nanotech, Technical University of Denmark, Building 345B, DK-2800 Kgs., Lyngby, Denmark. Electronic address: mikkel.hansen@nanotech.dtu.dk.
authors.forename: Mikkel F
authors.initials: MF
authors.lastname: Hansen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D060328:Magnetic Phenomena; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction; D019809:Streptavidin / Q000737:chemistry
chemical_list: D058185:Magnetite Nanoparticles; D004247:DNA; D019809:Streptavidin
keywords: DNA; DNA denaturation; GMR biosensor; Hybridization assay; Magnetic Biosensor; PCR