An angle-scanning Kretschmann configuration SPR instrument allowing multiplexed analysis is presented. Laser light was guided through optics that converted the collimated light into a line-shaped beam, which was directed to a prism, illuminating the gold sensor surface over a 1 × 10 mm area. The reflected light was led to a CCD detector providing simultaneous readout of individual analysis spots along the laser line at a selected angle (fixed-angle detection) or in scanning-angle mode (width of 35°). Full SPR curve could be measured every 3.6 s for each illuminated spot on the sensor surface. Two in-house manufactured flow cell designs were used for evaluating multiplexed angular-scanning SPR. The first comprised six parallel channels with the laser line perpendicular to the flow direction in order to allow interrogation of the sensor surface in the six channels. Refractive index changes by varying solution composition, and adsorption of different concentrations of albumin to the sensor surface could be correctly monitored simultaneously in each of the channels. In the second flow-cell design the laser line was coinciding with the flow path, allowing recording of SPR curves along a 10-mm length of the sensor surface. Adsorption of layers of positively and negatively charged polyelectrolytes could be consistently measured for sixteen selected positions along the channel. As a proof of principle, several target proteins were immobilized on different positions along the sensor and the binding of various antibodies with these proteins was monitored simultaneously, showing excellent selectivity and reproducibility for probing antibody-protein interactions in a multiplexed fashion.
- Angular-scanning SPR
- Antibody-protein interaction