The work described in this thesis is to develop a microscope into a high resolution bio-molecular interaction sensor. A "prism less" widefield surface plasmon microscope has been constructed and applied to imaging of interactions of protein and its antibody in aqueous media through a high NA objective. There are two main parts in this thesis: (1) design and layout of a high resolution angle scanning widefield surface plasmon resonance microscope; and (2) the application to bio-molecular interactions. In the first part, an angle-scanning widefield surface plasmon imaging (AW-SPRI) system consisting of an optical system, a liquid handling system and a data processing system is described. In the optical system, surface plasmons are excited by objective coupling. A spatial light modulator in a conjugate back focal plane of the objective lens allows dynamic control of illumination angle. The reflected bright-field widefield images, encoded with SPR signals, are detected by a CCD. The SPR signals in the images are decoded by a signal processing algorithm. AW-SPRI also combines well-controlled liquid handling units in order to monitor bio-molecular interactions or detect analytes in water-based solvent. The system shows high sensor resolution ( 5 x 10-5 RIU) as a biosensor. In addition, the edge response of A W -SPRI images with the BSA grating vector parallel to the incident polarization direction is 6.5 flm in air and 7.6 urn in water and the edge response with the BSA grating vector perpendicular to the incident polarization direction is 4.3 urn in air and 4.8 urn in water. The system presents high spatial resolution, too. The second part introduces sensor chip preparation and bio-molecular interactions. The sensor chip is where the bio-molecular interaction takes place and where the biochemical binding event is transduced into SPR signals. There are three types of sensor chips mentioned in this thesis which are bare gold-coated coverslips, protein grating patterns on a gold surface created by micro-contact printing, and protein grating patterns covalently immobilised on a gold surface created by photolithography. The patterned proteins on gold surfaces as our sensor chips are used to perform bio-molecular interactions. The results show our system can be used for comparison or determination of the concentration of ligands on the sensor chip or the affinities of the analyte with different samples on sensor chips. In addition, the measurement of affinity and rate constants show that the AW-SPRI can be used to measuring binding processes and carry out kinetic analysis of macromolecular interactions with standard interaction cycle method. Although the errors are larger than with the commercial SPR machines (SR 7000DC), they are nevertheless of similar order of magnitudes. To the authors' knowledge, this is the first demonstration of such high spatial resolution for quantitative, label-free, real-time detection of bio-molecule interaction cycles.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:556099 |
| Date | January 2011 |
| Creators | Tan, Han-Min |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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