| The potentiometric responses of dinonylnaphthalenesulfonate (DNNS)-based polymer membrane electrodes toward a variety of synthetic polycation peptides with different amino acid sequences and compositions were explored. Both the equilibrium and non-equilibrium EMF changes of polycation sensitive electrodes were found to depend on the structural characteristics of the given polycations, including the hydrophobicity, and charge distributions.; Two novel homogeneous binding/immunoassays were developed using polycation sensitive membrane electrode detection. One of the assays utilized synthetic polycations as labels. Theophylline was chosen as the model analyte and a synthetic peptide rich in arginine residues was employed as the label. Results demonstrated that sub-micromolar levels of theophylline could be detected potentiometrically without any need for discrete separation or washing steps. The second approach for using polyion sensors as detectors for devising homogeneous immuno/binding assays was based on use of trypsin as a label and protamine served as a substrate for a trypsin-analyte conjugate. In the presence of a binding protein, the activity of the trypsin-analyte conjugate was shown to be inhibited. Activity, as measured with the electrode detector, was shown to be regained in proportion to the amount of free analyte in the sample. Nano molar detection limits for model analyte biotin were achieved using this approach.; Beyond electrochemical detection of polycations, it was demonstrated that microtiter plates modified with thin, dichlorofluorescein octadecyl ester (DCFOE)-doped polymer films respond to polycations via an unusual response mechanism. The response was ascribed to the formation of a colloidal phase at the film/sample interface, resulting from extraction of DCFOE in deprotonated form to create a micelle ion-pair complex with the polycation species. In the microtiter plate arrangement, it was shown that such films could be used to detect protamine and other large polycation structures at concentrations ranging from 5--100 mug/mL. The polycation response was reproducible and was achieved over a reasonable time period of equilibration (10 min).; The microtiter plate modified with the DCFOE-based optical sensing film was proven useful for detecting polycations such as protamine in both buffer and plasma. It was further shown that such a detection mode could be utilized to quantitate heparin levels in plasma via protamine titrations and monitor specific protease activities (trypsin and plasmin). The ability to use such a technology to quantitate low levels of plasmin activity enabled the same microtiter plate detection system to be employed in the design of novel assays for monitoring specific plasminogen activators (e.g., urokinase, streptokinase, etc.) at therapeutic levels in plasma samples. |
