The use of ionomer membranes to enhance the selectivity of potentiometric enzyme electrodes in flow-injection measurement arrangements is examined. The ionomer membranes employed are permeable to analyte substrates but relatively impermeable to detectable ions that would normally interfere with the measurement of the substrates if the enzyme electrodes were in direct contact with the sample. As a model system, the selectivity of enzyme electrodes prepared with nonactin-based ammonium-sensitive polymeric membranes is evaluated. In the preferred configuration, a thin hydrophilic anion-exchange membrane is incorporated within a flow-through dialysis unit upstream from the enzyme-electrode detector. As the sample passes through the dialysis unit, neutral or anionic analyte molecules (urea or glutamine) move through the membrane while the permeation of endogenous ammonium ions and other cations in the sample is retarded. A flowing recipient buffer on the other side of the membrane carries the analyte substrate to the enzyme-electrode detector. Enhancements in selectivity for analyte substrates over endogenous ammonium and potassium ions are greater than or equal to 9-fold when compared to enzyme-electrode flow-injection analysis (FIA) systems assembled without the ionomer membrane unit. The analytical utility of the proposed system is demonstrated by the accurate measurements of urea in blood serum and L-glutamine in hybridoma bioreactor media.

A new solid-phase enzyme-linked competitive binding assay for vitamin B12 (cyanocobalamin) is described. The assay is based on the competition between analyte B12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B12 conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solidphase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 3 x 10-1~ x 10-8 M (using 100 #1 sample) with high selectivity over other biological molecules.

A novel approach for eliminating positive errors from endogenous ionic interferences when using ion-selective electrodes as detectors in flow-injection enzyme-based blosensing configurations is described. The method involves using a high background level of interfering ions in the sample diluent/carrier stream to convert the normally logarithmic potentiometric sensor into a linear detector over a given concentration range of primary ions. A split-stream single-detector arrangement provides a convenient means to compensate for varying levels of background interferent ions in the injected samples. One portion of the split stream passes directly to the ion-electrode detector, yielding a signal linearly related to the concentration of endogenous primary ions in the sample. The second portion of the split sample is delayed while passing through an immobilized enzyme that generates electrode detectable primary ions in proportion to the concentration of the substrate analyte in the sample. Two linear equations with two unknowns describe the twin potentiometric responses observed. The concept is demonstrated by the accurate determination of L-glutamine in hybridoma bioreactor media via the use of an ammonium-ion-selective membrane electrode detector and immobilized glutaminase enzyme.

A new approach for reducing poslttve errors caused by endogenous catlomc Interferences when usmg ammonmm ion-selectwe electropes as detectors m unmoblllzed enzyme-based flow-mlectlon analysis systems IS described The method mvolves the use of an m-hne tubular catlon-exchange unit (e g , Nafion) placed between the mjection valve and the downstream munoblhzed enzyme reactor/electrode detector portlon of the system Interferent catlon species wlthm the sample slug are exchanged for other cattons (replacement tons, e g, LI+) contamed wlthm a reservOlr solution surrounding the Ion-exchange tubmg The membrane electrode ehblts much less response toward the replacement cations, consequently, the detected concentration of ammomum ions generated downstream wlthm the enzyme reactor IS directly proportlonal to the level of analyte substrate present m the sample The Influence of various experlmental parameters on the efficiency of the m-hne exchanger as well as the general advantages and hnutatlons of this approach are exammed The analytical utlhty of the concept IS demonstrated by the rapld and accurate determmatlon of r-glutamme m bloreactor media via the use of lmmoblhzed glutammase enzyme