Immunomagnetic concentration of antigens and detection based on a scanning force microscopic immunoassay

Scanning Force Microscopy has already been shown to be a convenient and rapid method for sensitive antigen detection and quantification. Here, we describe different improvement steps brought to a TSH Scanning Force Microscopic ImmunoAssay (SFMIA), each of them aiming to solve a previous limitation of the solid phase test format and leading to a significant sensitivity enhancement. First, superparamagnetic nanoparticles conjugated to monoclonal anti alphaTSH antibodies were used for the specific TSH capture step. Their magnetic properties allowed easy separation of the complexes obtained from relatively large reaction volumes by application of a High Gradient Magnetic Field System. As a consequence, complex formation could proceed in a stirred solution, which greatly enhances binding rates compared to previous 'static' conditions of solid-phase reactions. It was established that, despite their small size, magnetic complexes could be moved over short distances by a NdFeB magnet magnetic field. This property was exploited to overcome diffusion barrier and boundary layer constraints and to drive magnetic complexes through the liquid, towards anti-betaTSH antibodies immobilized on silica wafers. Finally, we significantly increased the complex number/surface area by a stepwise reduction of the biospecific solid phase area. The proposed steps permitted a 3-fold improvement in the TSH SFMIA dynamic range. Moreover, as little as 0.02 pg/ml (0.1 nIU/ml or 0.8 amol/ml) of TSH could be detected using 1 ml sample volumes. This is over 100 times more sensitive than the current performance of commercialized automated systems.