Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma

Two different fibrinogen (Fib) amperometric immunosensing designs based on the use of magnetic beads (MBs) and a novel specific nanobody (Nb) expressed in Escherichia coli are described for the first time. The immunological reaction for Fib detection was performed on COOH-MBs or His-Tag-Isolation-MBs as solid support for the immobilization of the antigen or the captured Nb. Direct and indirect competitive magnetoimmunosensing configurations have been tested and compared. In the former one, Fib and biotinylated Fib competed for the immobilized Nb binding sites while the latter configuration involved competition of free Fib in solution and immobilized Fib for binding to a fixed amount of the specific biotinylated Nb. Labeling of the captured biotinylated Nb or antigen was made with streptavidin-HRP. The modified magnetic beads were captured by a neodymium magnet on the surface of screen-printed carbon electrodes (SPCEs). Amperometric detection was accomplished at -0.20 V (vs. Ag pseudo-reference electrode) by measuring the catalytic current arising upon addition of H2O2 and using hydroquinone (HQ) as redox mediator in solution. A better analytical performance was achieved with the indirect competitive immunoassay with a detection limit of 0.044 μg mL(-1) Fib. The usefulness of both approaches was successfully demonstrated by analyzing an international standard for Fib plasma. The assays could be carried out in diluted plasma samples in a total analysis time of 90 min.