Planar Waveguides as High-Performance Sensing Platforms for Fluorescence-Based Multiplexed Oligonucleotide Hybridization Assays

16-Mer and 22-mer oligonucleotide capture probes with an amino function at the 5‘-end were covalently immobilized on (3-glycidoxypropyl)trimethoxysilane (GOPTS) silanized planar waveguides in “checkerboard”-style 2-dimensional arrays by means of ink-jet printing in order to demonstrate the potential of multiplexed planar waveguide biosensing. The laser-induced fluorescence was collected by a CCD camera, providing quantitative information regarding the intensity of individual sensing pads. In model hybridization assays with Cy5-labeled complementary oligonucleotides, sample concentrations down to 50 fM were successfully detected within 12 min. The overall spot-to-spot variability of the ink-jet immobilization in combination with amino functionalized oligonucleotides and GOPTS silanized waveguides was found to be about 12% and almost independent of the formation of an equilibrium between the labeled analyte and capture probes. Both pipet-made and ink-jet-made oligonucleotide spots showed comparable assay performances.