Pinched flow fractionation devices for detection of single nucleotide polymorphisms

TY - JOUR

T1 - Pinched flow fractionation devices for detection of single nucleotide polymorphisms

AU - Larsen, A.V.

AU - Poulsen, L.

AU - Birgens, H.

AU - Dufva, M.

AU - Kristensen, A.

N1 - Times Cited: 0ArticleEnglishKristensen, ATech Univ Denmark, Nanotech Dept Micro & Nanotechnol, Bldg 345 E,Oersted Plads, DK-2800 Lyngby, DenmarkCited References Count: 22292OTROYAL SOC CHEMISTRYTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDCAMBRIDGE

PY - 2008

Y1 - 2008

N2 - We demonstrate a new and flexible micro fluidic based method for genotyping single nucleotide polymorphisms ( SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a micro fluidic pinched flow fractionation (PFF) device. The micro fluidic PFF devices with 13 mu m deep channels were fabricated by thermal nanoimprint lithography ( NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 mu m and 5.6 mu m were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs Udgivelsesdato: 2008

AB - We demonstrate a new and flexible micro fluidic based method for genotyping single nucleotide polymorphisms ( SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a micro fluidic pinched flow fractionation (PFF) device. The micro fluidic PFF devices with 13 mu m deep channels were fabricated by thermal nanoimprint lithography ( NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 mu m and 5.6 mu m were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs Udgivelsesdato: 2008

M3 - Journal article

SN - 1473-0197

VL - 8

SP - 818

EP - 821

JO - Lab on a Chip

JF - Lab on a Chip

IS - 5

ER -