New dual ionophore immunosensor concept: modeling and experimental support

Sarah Jean Haarsma

Literature shows that ion-selective electrodes (ISEs) can be modified to create a potentiometric immunoassay by covalent attachment of a hapten to the ionophore and use of immunocompetition to modulate transmembrane ion flux. Nernstian theory applied to these immunoassays predicts constant voltages dependent only on the transmembrane concentration gradient so the origin of the changes described in the literature is unclear. The potential decreases may be attributable to the inability to measure the true voltages as a result of reduced currents through the membrane. In this thesis I present a manuscript prepared for publication in Biosensors and Bioelectronics, which proposes use of a dual ionophore system (di-ISE) with opposing Na+ and K+ concentration gradients. Advantages of di-ISEs are two-fold: 1) the second ionophore provides sufficient base current even on capture of the primary ion by antibody; and 2) opposing ion gradients allow voltage modulation between the oppositely charged equilibrium potentials of the two ions. Models predict accompanying voltage changes as high as 60 mV for an ISE membrane containing 10% K+ ionophore valinomycin, and 1/10th the molar amount of Na+ ionophore. This will occur when K+ ionophore availability reduces from 100% to 10%. Predictions show this voltage change will be reduced by up to 60% when including the Na+ selectivity coefficients for the K+ carrier 4'aminobenzo-18-crown-6 of 0.090, and K+ selectivity coefficient of Na+ ionophore X (NaX) of 0.035. However, one can compensate for voltage reduction by reducing the NaX concentration or the Na+ gradient. Preliminary data are shown for antibody attachment in an ISE containing a dinitrophenol hapten and a di-ISE with cardiac troponin I hapten. As there is a growing need for efficient biosensors for organic species and proteins, further characterization of this type of immunosensor could lead to quick, simple immunosensors for low-level testing of organic molecules and proteins. One example of this is the monitoring of cardiac health through testing of the low-abundance cardiac troponin isoforms which are present in the circulating blood.

New dual ionophore immunosensor concept: modeling and experimental support

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