Kinetics of the competitive response of receptors immobilised to ion-channels which have been incorporated into a tethered bilayer

A competitive ion channel switch (ICS) biosensor has been modelled yielding ligand mediated monomer-dimer reaction kinetics of gramicidin (gA) ion-cha nnels within a tethered bilayer lipid membrane. Through employing gramicidi n A, functionalised with the water-soluble hapten digoxigenin, it is possib le to cross-link gramicidin to antibody fragments tethered at the membrane/ aqueous interface. The change in ionic conductivity of the channel dimers m ay then be used to measure the binding kinetics of hapten-protein interacti ons at the membrane surface. The approach involves measuring the time depen dence of the increase in impedance following the addition of a biotinylated antibody fragment (b-Fab'), which cross-links the functionalised gramicidi n monomers in the outer layer of the lipid bilayer to tethered membrane spa nning lipid. The subsequent addition of the small molecule digoxin, (M, 781 Dal, competes with and reverses this interaction. The model provides a quantitative description of the response to both the c ross-linking following the addition of the b-Fab' and the competitive displ acement of the hapten by a water-soluble small analyte. Good agreement is o btained with independent measures of the cross-linking reaction rates of th e gramicidin monomer-dimer and the b-Fab : hapten complex. The rate and amp litude of the competitive response is dependent on concentration and provid es a fast and sensitive detection technique. Estimates are made of the concentration of gramicidin monomers in both the inner and outer monolayer leaflets of the membrane. This is used in the cal culation of the gramicidin monomer/dimer equilibrium constant, K-2D3. Other considerations include the membrane impedance limit set by the membrane le akage which is also a function of the concentration of the gA monomer conce ntration, and the two-dimensional kinetic association constant k(2D2), of t he hapten : b-Fab' complex. The gA dimer concentration is dependent on both the concentration of ga-dig and of the tethered streptavidin : b-Fab' comp lexes. The model shows that the 2D dissociation constant k(2D3)(-1), must be at le ast 10 times faster than the 3D dissociation constant k(3D2)(-1), for digox in to completely reverse the cross-linked hapten-receptor interaction at th e membrane interface.