An improved polycation-sensitive membrane electrode based on the cation exchanger dinonylnaphthalenesulfonate (DNNS) has been developed. The electrode yields a significant potentiometric response (>50 mV) towards micromolar levels of protamine and other polycations in samples as complex as whole blood. The EMF response characteristics of this electrode are consistent with the non-equilibrium steady-state response mechanism previously developed for polyanion-sensitive electrodes. The DNNS-based electrode serves as a sensitive end-point detector for measuring heparin levels (0--10 U/mL) via potentiometric titrations with protamine. Heparin concentrations measured in whole blood using the electrode correlate well with other techniques for measuring heparin levels in samples obtained from patients undergoing bypass surgery. More rapid heparin measurements can be achieved by employing a continuous infusion of protamine into the sample. By suitably modifying the membrane composition, the protamine titration method can also be applied to the determination of low molecular weight heparins (LMWHs) in whole blood. It is shown that the nature of the LMWH determines its binding affinity to protamine, thus governing its ability to be determined via manual protamine titrations. Finally, these electrodes are further used as detectors for measuring the activity of proteases by following the degradation of polycationic substrates. By employing synthetic arginine-rich peptides that contain amino acid residues specifically cleaved by the target protease, more specific assays can be developed. It is also demonstrated that this assay methodology can be applied to measure protease inhibitors (e.g., aprotinin) in buffered and treated plasma samples.Ph.D.Analytical chemistryApplied SciencesBiomedical engineeringPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131764/2/9929931.pd
