Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument.

We present a new optical biosensor for probing molecular binding to a water/glass interface. The system is designed to measure the kinetics of surface reactions down to low analyte concentrations straightforwardly. The selective detection of surface bound fluorescence is achieved by collecting supercritical angle fluorescence (SAF) emission of surface bound molecules into the glass. Thereby the expansion of the detection volume into the aqueous probe is reduced to about one sixth of the fluorescence wavelength, consequently bulk fluorescence from the solution is rejected successfully. The SAF-signal is captured by a parabolic glass lens, which leads to high spatial collection efficiency and detection sensitivity. The sensor has an inverted optical design and is compatible with common glass cover slips, which strongly facilitates operation for the user working in the biological and biochemical fields. The performance of the system is demonstrated by real time measurements of antibody-antigen reactions. Rate constants of the reaction were extracted. Antigen concentrations were detected down to 10(-13) mol/l.