Biomolecular sensing using wavelength-scanning reflective interferometry (ws-RI)

Many sensing methods currently available for the detection of biomolecules such as oligonucleotides and proteins often require modification of the target analytes and need complex, expensive readout systems. An innovative chip-based detection technique for label-free biomolecular sensing, wavelength-scanning Reflective Interferometry (ws-RI) will be reported in this thesis. ws-RI is able to detect adsorption of materials on the chip surface at sub-monolayer scales with sensitivity to average adsorption below 0.2 nm. Change of topology on the substrate that results from binding of target molecules to complementary probe molecules immobilized on the sensing surface can be detected by ws-RI and ws-RI can also give a quantitative estimate for the amount of bound targets.; ws-RI is based on removal of the destructive interference in the reflected intensity from an anti-reflection coated silicon substrate at a certain incidence angle. Upon binding of target molecules the reflectivity from the substrate surface will significantly increase and the contrast can be easily measured. The silicon substrate surface was prepared with a hydrophilic pattern to make arrays of reaction wells for parallel detection. We successfully detected hybridization of 40 fmol/mm2 short, label-free DNAs (equivalent to the amount in 2mul 20nM DNA solution) with negligible nonspecific binding. We also succeeded in detecting even smaller amounts of short label-free DNAs via neutral complementary PNA probes. Signal amplification using positively charged gold nanoparticles to electrostatically decorate the chip after PNA/DNA hybridization further improved the detection limit of ws-RI to 2 fmol/mm 2 (equivalent to the amount in 0.5mul 4nM DNA solution). Detection of human-alpha-thrombin using an immobilized DNA aptamer on the chip was also done, illustrating the potential utility of ws-RI for proteomic screening. The simplicity of the ws-RI should make it useful for a wide selection of target molecules where suitable molecular recognition chemistry is available.; We also present a theoretical treatment of ws-RI showing how sensitivity is limited by factors such as bandwidth and angular divergence of the probe beam and substrate oxide roughness. Further improvements in sensitivity can be expected with flatter oxides and with lasers having better collimation and monochromaticity as probe sources.