Sensitivity enhancement of optical immunosensors by the use of a surface plasmon resonance fluoroimmunoassay

Optical immunosensors employing evanescent wave techniques have the potential to address the requirements of the ‘alternative site’ market; however, this potential has yet to be realised. The development of ‘direct’ sensors, such as those using surface plasmon resonance (SPR), has been hampered by problems of non-specific binding and poor sensitivity to small molecules. ‘Indirect’ sensors (for example, those employing a fluorescently labelled reagent) overcome many of the problems of direct sensors but require more sophisticated instrumentation because of the low light levels detected. In an attempt to combine the best features of the two techniques, an indirect SPR fluoroimmunoassay (SPRF) technique has been investigated. The surface field intensity enhancement produced by SPR is used to boost the emission from a fluorescently labelled immunoassay complex at a metal surface. The potential of the method is demonstrated by assaying for human Chorionic Gonadotrophin (hCG) in serum. Enhanced sensitivity over conventional total internal reflection fluorescence (TIRF) and SPR techniques was achieved.