title: A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.
abstract: Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Mayr
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Haider
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Thünauer
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselgrübler
authors.affiliation: Institute of Applied Physics, Vienna University of Technology, 1040 Vienna, Austria.
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria. Electronic address: jan.hesse@cbl.at.
authors.forename: Jan
authors.initials: J
authors.lastname: Hesse
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005453:Fluorescence; D057230:Limit of Detection; D044085:Microfluidics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic
chemical_list: D012313:RNA; D004247:DNA
keywords: DNA; Lab-on-a-chip; Microarray; Microfluidic biochip; RNA; Ultra-sensitive detection