Interferometric immunoassay in a FIA-system: a sensitive and rapid approach in label-free immunosensing

Optical measurement principles gather growing attention in the field of immunosensors. Label-free systems for online monitoring are of special interest with respect to speed and cost. Recently we have shown that spectral interferometry allows online monitoring of immunoreactions. In an assay for IgG, using a batch setup, concentrations from 1 μg ml^-1 to 25 μg ml^-1 IgG can be determined within an incubation time interval of about two minutes. In this paper we report results using a FIA-system. Determinations of IgG were performed from continuously flowing sample solution and limited sample volumes pumped with alternating flow directions over the sensor surface. The detection limit for the given system is in the range of 0·1 μg ml^-1 IgG. Sample volumes could be reduced to 200 μl with an active sensor surface of about 30 mm², 0·5 mm depth and about 15 μl volume of the flow cell. Immunoreactions were carried out on thin films of synthetic silica on high refractive index glass substrates. Interferograms were recorded with a diode array spectrometer and stored for offline evaluation on a personal computer connected to the spectrometer. Binding curves were evaluated by nonlinear least squares regression. Initial reaction rates, computed from fit results, were used for the determination of concentrations. For the measurement of nonspecific binding effects on immunoassay signals, differently pretreated substrates were incubated with samples of 10% calf serum. The feasibility and the limitations of spectral interferometry for affinity sensing, characterization of immunoassays and for immunosensor development are discussed.