A label-free continuous total-internal-reflection-fluorescence-based immunosensor

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (Kd > 10−6 M), fast kinetics (koff > 1 s−1), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1–5 μM for panose, and 10–15 μM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of ΔH° and ΔS° from the temperature dependence of Kd indicated an enthalpic driven antigen–antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.