A rotating electrode configuration is evaluated as a means to lower the detection limits of newly devised polyion-sensitive membrane electrodes (PSEs). Planar potentiometric polycation and polyanion PSEs are prepared by incorporating tridodecylmethylammonium chloride and calcium dinonylnaphthalenesulfonate, respectively, into plasticized PVC or polyurethane membranes and mounting disks of such films on an electrode body housed in a conventional rotating disk electrode apparatus. Rotation of the PSEs at 5000 rpm results in an enhancement in the detection limits toward heparin (polyanion) and protamine (polycation) of at least 1 order of magnitude (to 0.01 unit/mL for heparin; 0.02 microg/mL for protamine) over that observed when the EMF responses of the same electrodes are assessed using a stir-bar to achieve convective mass transport. A linear relationship between omega(-1/2), where omega is the rotating angular frequency, and C1/2, the polyion concentration corresponding to half the total maximum deltaEMF response toward the polyion species, is observed. It is further shown that the rotating polycation sensor can be used as an end-point detector to greatly enhance (relative to nonrotated indicator electrode) the analytical resolution and precision for measurement of low concentrations of heparin when such samples are titrated with protamine. The theoretical basis for lowering the detection limits by rotating PSEs is discussed based on the unique nonequilibrium response mechanism of such sensors.

Microtiter plate wells modified with thin (approximately 20 microm) polymeric films capable of optically sensing macromolecular protamine and other polycationic species are described. The plates are prepared by coating the bottom of each well of a conventional 96-well polypropylene plate with an adherent polymer film (a mixture of poly(vinyl chloride) and polyurethane) containing a lipophilic 2',7'-dichlorofluorescein derivative. Surprisingly, optical response toward polycations is shown to result from the extraction of the fluorescein derivative from the polymer film into a lyophobic colloidal phase at the sample/film interface. This new phase is likely composed of a micellular-type ion pair complex between the analyte polycation from aqueous sample phase and the deprotonated form of the fluorescein derivative. Accumulation of the deprotonated fluorescein species in this interfacial region induces an absorbance change measured at 540 nm. Optimized plates can be used to sense protamine concentrations in the range of 0-100 microg/mL in 10 min with little or no response to physiological levels of common cationic species (Na+, K+, Ca2+, etc.). The modified plates are shown to be useful as simple optical detectors for measuring heparin levels in plasma via titrations with protamine and for monitoring protease activities (trypsin and plasmin) that cleave polycationic peptides/proteins such as protamine into smaller peptide fragments that are not detected by the sensing films. Assays for "clot busting" plasminogen activators (streptokinase, urokinase, and tissue plasminogen activator) are also demonstrated using this relatively simple microtiter plate-based polycation detection system.

The microstructure of plasticized PVC membranes in the dry state and during the process of soaking in heavy water has been studied by small-angle neutron scattering. In the dry membrane, inhomogeneities were found. The membrane structure is well described by a polydisperse hard-sphere model. The mean diameter of the dispersed spherical inhomogeneities is ∼6 nm, which is smaller than the estimated dimension of a single statistically curled PVC polymer chain in the membrane. The values of the best-fit parameters and their change with membrane composition suggest that the particles consist of unplasticized PVC, probably in the crystalline state. The type of plasticizer, the plasticizer content, and the addition of a lipophilic salt were found to influence the water uptake significantly. Water uptake did not change the microstructure due to the original (i.e., dry state) inhomogeneities in the membranes.

No abstract