Elimination of endogenous cationic interferences in potentiometric flow injection biosensing systems.

Abstract

Two new approaches were examined in an effort to reduce or eliminate endogenous cation interference problems that have limited the practical use of potentiometric ion/gas detectors in enzyme-based biosensing systems. These generic approaches are applied specifically in the design of flow-injection analysis systems suitable for the direct determination of L-glutamine in bioreactor media. The first approach involves the use of a cation-exchanger tubular membrane in which interferent ions are replaced by cations that do not interfere with potentiometric measurements. This method is suitable for the determination of negatively charged analytes which cannot permeate the tubing. In the second approach, an anion-exchange membrane is incorporated into a dialysis unit within a flow-injection analysis (FIA) system. Here, endogenous cationic interferences are repelled by the ionomer membrane, while the analyte species (glutamine) is transported from the sample stream across the membrane to a flowing recipient stream that passes by a potentiometric enzyme-electrode detector. This approach is useful for detecting neutral or negatively charged analytes. L-Glutamine is chosen as the target model analyte for the evaluation of these two approaches owing to its important role as an essential energy source in mammalian cell cultures, including monoclonal antibody producing hybridoma cells. Under optimized conditions, both the cation and anion-exchange membrane-based methods provide satisfactory reduction from cationic interferences in media samples. Indeed it is shown that these methods can be utilized to design FIA systems that offer adequate detection limits (down to 0.2 mM) and selectivities for direct measurements of L-glutamine in undiluted hybridoma cell media samples. In the final FIA biosensing systems the enzyme glutaminase was immobilized on nylon tubing, on controlled pore glass (CPG) beads, and finally on the surface of an ammonium ion-selective electrode (ISE). Enzyme-CPG reactors offered a higher substrate conversion efficiency than the other two arrangements.