Optimization of antibody immobilization for sensing using piezoelectrically excited-millimeter-sized cantilever (PEMC) sensors

The effectiveness of antibody immobilization on sensor performance was evaluated using a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor and a model protein, bovine serum albumin (BSA). The immobilization parameters—antibody and activation reagent concentration and reaction time were systematically varied and the resultant sensor response to 1 pg/mL BSA was measured. ghest frequency shift due to BSA attachment (1931 ± 60 Hz) was obtained when 100 μg/mL antibody solution was activated with EDC (2 mM) and sulfo-NHS (5 mM) for 30 min prior to reaction with primary amine on the sensor surface. Increasing activation time from 30 to 60 min resulted in an 8% decrease in sensor response, while activation at 0.2 mM EDC and 0.5 mM NHS resulted in a 65% decrease. The logarithmic correlation between antibody concentration and sensor response suggests that a lower antibody concentration of 10 μg/mL is sufficient for BSA at 1 pg/mL. With 10 μg/mL antibody, the PEMC sensor response was 725 ± 50 Hz (n = 2) for 1 pg/mL BSA with a signal to noise ratio of 41.