Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery

A potentiometric biosensor for determination of trypsin is described based on current-controlled reagent delivery. A polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation exchanger is used for in site generation of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which the protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5 U mL^-1 with a detection limit of 0.3 U mL^-1. The proposed detection strategy provides a rapid and reagentless way for detection of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels.