Improved direct piezoelectric biosensors operating in liquid solution for the competitive label-free immunoassay of 2,4-dichlorophenoxyacetic acid

The development of a label-free direct piezoelectric immunosensor is reported. The piezoelectric crystals were modified with 2,4-dichlorophenoxyacetic acid (2,4-D) using coupling procedures based on self-assembled monolayers of suitable thiocompounds (aminothiophenol, cysteamine, cystine and dithiobis(succinimidyl)propionate) on the surface of gold electrodes of the crystals. The piezoelectric biosensor has been placed in a flow-through cell and the affinity binding of two monoclonal antibodies against 2,4-D on the modified piezoelectric crystals was studied in real time without any additional labels. For the interactions, the kinetic equilibrium association constants K-a were 12.6 and 25.0 mu M-1 for the antibodies E2/G2 and E4/C2, respectively. The system was used for competitive determination of free 2,4-D in water. For the antibodies E2/G2 and E4/C2 (both in concentration of 15 mu g/ml), the limits of detection (10% decrease of relative binding of the antibody) for free 2,4-D were 0.27 and 0.24 mu g/l, respectively. The total time for one measurement was below 25 min (10 min preincubation of antibody with sample, 10 min binding reaction, 5 min regeneration).