Construction and characterization of the direct piezoelectric immunosensor for atrazine operating in solution.

The direct immunosensor for determination of the herbicide atrazine was studied. The gold electrodes of the piezoelectric quartz crystal were silanized and activated using glutaraldehyde. The bioaffinity ligand atrazine was linked through albumin as a spacer molecule. The modified piezoelectric crystal was placed in a flow cell and all measurements were performed directly in flowing solution. The interaction of the anti atrazine monoclonal antibody (MAb, clone D6F3) with the immobilized atrazine was characterized using both crude ascitic fluid and Protein A-purified MAb preparates. The association and dissociation rate constants were determined, ka = 1.21 x 10(5) M-1S-1 and kd = 4.0 x 10(-4)S-1. The competitive determination of free atrazine was studied using different dilutions (100x, 250x and 1000x) of the ascitic fluid containing MAb. MAb was preincubated with atrazine (concentrations 0-1 microgram/l) for 15 min and the mixture was then introduced to the flow cell. As a signal, either the rate of frequency decrease or the relative change of frequency after the fixed binding period (10 min) was evaluated. As expected, the higher dilutions of MAb provided improved sensitivity for the analyte. For the 1000x diluted ascitic fluid, 0.1 and 1 microgram/l atrazine caused 5 and 30% decreases of the relative binding of MAb, respectively. Repeated use of the crystals was achieved using a 5 min flow of 100 mM NaOH for regeneration. The results obtained seem to be promising for determination of atrazine in drinking water using direct piezoelectric immunosensors.