Innovative surface characterization techniques applied to immunosensor elaboration and test: Comparing the efficiency of Fourier transform–surface plasmon resonance, quartz crystal microbalance with dissipation measurements, and polarization modulation–reflection absorption infrared spectroscopy

Three sensitive and original transduction techniques have been used to monitor the immobilization of anti-rabbit immunoglobulins (anti-rIgGs) and the detection of rIgGs on gold transducers. Polarization modulation–reflection absorption infrared spectroscopy (PM–RAIRS), quartz crystal microbalance with dissipation measurements (QCM–D), and Fourier transform–surface plasmon resonance (FT–SPR) were combined to achieve the best sensitivity and a large dynamic range in the target detection step. Their performances were compared after having checked that the layers adsorbed on the three different gold substrates were identical. The studied immunosensors were elaborated by building a thiolamine layer on gold surface, followed by its derivatization by glutaraldehyde and covalent binding of a monoclonal secondary IgG. The antibody attachment step was monitored in a wide range of concentrations (1–50 μg/ml). Then the built immunosensors were used to detect the rIgG recognition. PM–RAIRS analyses, performed under air, supplied ex situ data, whereas FT–SPR and QCM techniques were used in situ, enabling on-line detection of recognition processes. Interestingly, the three techniques suggested that the antibody coverage gets saturated for approximately 20 μg/ml in solution. In the very low concentration range (1 μg/ml), antibody binding was detected by the three techniques, but FT–SPR leads to an intense signal with a wavenumber shift of approximately 30 cm⁻¹; one may expect, by FT–SPR, a detection limit of the order of a few tenths of μg/ml. Ongoing experiments aim at determining the limit of detection and dynamic range of the very promising FT–SPR technique.