Immunosensor based on total internal reflection fluorescence principle

The antigen (human-IgG) was covalently attached to the surface of a planar quartz waveguide. When the light beam passes through the waveguide, only the FITC labelled antibody primarily bound (or very close) to the surface was excited by the evanescent wave on the surface, which resulted in spatial separation of the bound Ag-Ab compounds from the free molecules in the bulk. The non-binding signal, non-specific and specific binding signals were measured respectively, and the immuno-competitive assay was described. It was shown that the dynamic detecting range can be adjusted. When the concentration of competitive reagent is 50μg/ml, detecting limit of the sensor was approximately 1μg/ml. But for direct immunoassay of labelled antibody, the sensitivity is about 0.5μg/ml. The recycle of solid phase protein showed no obvious loss of biological activity after use of 8 times. It is expected that this immunosensor will have wide applicability in both routine and research field.