title: Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry.
abstract: This system allows the high-throughput protein interaction analysis on microarrays. We apply the interference technology 1λ-imaging reflectometric interferometry (iRIf) as a label-free detection method and create microfluidic flow cells in microscope slide format for low reagent consumption and lab work compatibility. By now, most prominent for imaging label-free interaction analyses on microarrays are imaging surface plasmon resonance (SPR) methods, quartz crystal microbalance, or biolayer interferometry. SPR is sensitive against temperature drifts and suffers from plasmon crosstalk, and all systems lack array size (maximum 96 spots). Our detection system is robust against temperature drifts. Microarrays are analyzed with a spatial resolution of 7 µm and time resolution of ≤50 fps. System sensitivity is competitive, with random noise of <5 × 10-5and baseline drift of <3 × 10-6. Currently available spotting technologies limit array sizes to ~4 spots/mm2(1080 spots/array); our detection system would allow ~40 spots/mm2(10,800 spots/array). The microfluidic flow cells consist of structured PDMS inlays sealed by versatilely coated glass slides immobilizing the microarray. The injection protocol determines reagent volumes, priming rates, and flow cell temperatures for up to 44 reagents; volumes of ≤300 µL are validated. The system is validated physically by the biotinylated bovine serum albumin streptavidin assay and biochemically by thrombin aptamer interaction analysis, resulting in a KD of ~100 nM.
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Burger
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|2 Institute for Biology III, Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Rath
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Woehrle
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.
authors.forename: Philipp A
authors.initials: PA
authors.lastname: Meyer
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.
authors.forename: Nessim
authors.initials: N
authors.lastname: Ben Ammar
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|2 Institute for Biology III, Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Normann
authors.initials: N
authors.lastname: Kilb
authors.affiliation: 3 Laboratory for CPI, IMTEK, University of Freiburg, Freiburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brandstetter
authors.affiliation: 4 Biametrics GmbH, Tübingen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Pröll
authors.affiliation: 4 Biametrics GmbH, Tübingen, Germany.
authors.forename: Guenther
authors.initials: G
authors.lastname: Proll
authors.affiliation: 5 Laboratory for Sensors, IMTEK, University of Freiburg, Freiburg, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Urban
authors.affiliation: 1 Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|2 Institute for Biology III, Biology, University of Freiburg, Freiburg, Germany.|6 BIOSS-Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Guenter
authors.initials: G
authors.lastname: Roth
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D007368:Interferometry / Q000379:methods*; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D011506:Proteins; D012710:Serum Albumin, Bovine; D001710:Biotin; D019809:Streptavidin; D013917:Thrombin
keywords: high-throughput protein analysis; imaging label-free detection; microfluidic biosensor; reflectometric interferometry