Qu et al. (2021) Biosensors and Bioelectronics 192, 113549
We present an innovative multiplexing concept on a fiber optic surface plasmon resonance (FO-SPR) platform and demonstrate for the first time the simultaneous detection of two targets using the same FO sensor probe. Co (III)-NTA chemistry was used for oriented and stable co-immobilization of two different His6-tagged bioreceptors. T2C2 and MDTCS (i.e. fragments of the ADAMTS13 metalloprotease linked to the thrombotic thrombocytopenic purpura disorder) served as model system bioreceptors together with their respective targets (4B9 and II-1 antibodies). Gold nanoparticles were used here in an original way for discriminating the two targets in the same sample, in addition to their traditional signal amplification-role. After verifying the specificity of the selected model system, we studied the bioreceptor surface density and immobilization order. Innovative approach to lower the bioreceptor concentration below surface saturation resulted in an optimal detection of both targets, whereas the order of immobilization of the two bioreceptors did not give any significant difference. By sequentially immobilizing the T2C2 and MDTC bioreceptors, we established calibration curves in buffer and 100-fold diluted human blood plasma. This resulted in calculated limits of detection of 3.38 and 2.31 ng/mL in diluted plasma for 4B9 and II-1, respectively, indicating almost the same sensitivity as in buffer. Importantly, we also proved the applicability of the established calibration curves for quantifying the targets at random and more realistic ratios, directed by the design of experiments. This multiplexing study further expands the repertoire of applications on the FO-SPR biosensing platform, which together with its intrinsic features opens up great opportunities for diagnostics and life sciences.