AVS 50th International Symposium
    Biomaterial Interfaces Thursday Sessions
       Session BI-ThM

Paper BI-ThM9
Real-Time, Quantitative Surface Plasmon Microscopy Measurements of Protein Adsorption

Thursday, November 6, 2003, 11:00 am, Room 318/319

Session: Biosensors
Presenter: J.S. Shumaker-Parry, University of Washington, presently at the Max Planck Institute for Polymer Research, Germany
Authors: J.S. Shumaker-Parry, University of Washington, presently at the Max Planck Institute for Polymer Research, Germany
M.H. Zareie, University of Washington
C.T. Campbell, University of Washington
Correspondent: Click to Email
Surface plasmon resonance (SPR) spectroscopy has become a popular technique for measuring biomolecular interactions in real time with high sensitivity and without labels. SPR microscopy provides the same advantages as SPR spectroscopy with the added feature of using a CCD camera to image changes in reflected light intensity across a large area of a sensor surface simultaneously with good spatial resolution (~ 4 µm). Recently we have developed quantitative SPR microscopy methods for measurement of adsorption and desorption processes in real time based on monitoring changes in reflected intensity at a high contrast angle. For a small range of angles in a linear region of a SPR curve, reflectivity changes are proportional to effective refractive index changes near the sensor surface. By fixing the angle of measurement at a high contrast angle in such a linear region, refractive index changes may be monitored in real time by measuring reflectivity changes for pre-selected regions of a sensor surface. By extending methods used to quantitate SPR spectroscopy wavelength and angle shifts to changes in reflectivity measured by SPR microscopy, quantitative, real-time adsorption measurements are possible. We used these methods to measure adsorption of a DNA-binding protein to its DNA-binding site immobilized in a double-stranded DNA (dsDNA) array on a streptavidin linker layer to demonstrate the use of SPR microscopy for parallel, high-throughput array-based analysis. A major advantage of these array-based studies is the ability to use array elements without the DNA-binding site as reference regions to correct for non-specific adsorption and common refractive index changes. The real-time detection limit for fast time resolution measurements is less than ~8 x 10@super 6@ proteins per 200-µm array spot.