Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry.

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 × 10-5 and 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.