Modeling and experimental verification of the performance of TIRF-sensing systems for oligonucleotide microarrays based on bulk and integrated optical planar waveguides

The detection of hybridization events on oligonucleotide microarrays in real time can be performed, using the optical principle of total internal reflection fluorescence (TIRF). We have investigated and compared three TIRF-sensing configurations using two bulk and one integrated optical planar waveguide as transducer platforms for oligonucleotide microarrays, which have been brought in contact with flow cells. Based on the ray optics model, expressions were derived for the calculation of the intensity of the CCD-camera signal generated by solved fluorophores in the flow cell volume. A noise analysis was performed and expressions for the calculation of the detection limit of the surface fluorophore density were derived. With a bulk optical single total internal reflection configuration a detection limit of 3.74 molecules/μm2, with a bulk optical multiple total internal reflection configuration a detection limit of 1.83 molecules/μm2 and with the integrated optical waveguide (IOW) configuration a detection limit of 0.013 molecules/μm2 was numerically estimated based on background data of the bulk volume signal. The derived analytical expressions address the full system, including light source, optical waveguide and the detection unit and can serve as a tool for TIRF-system design.