The quantification of small molecules in aqueous solution by surface bound supramolecular host molecules is an important goal in the research field of chemo- and biosensor development. In this paper we present an attempt to quantify the interaction of different charged guest molecules with chemisorbed monothiolated β-cyclodextrin monolayers by means of impedance spectroscopy in the presence of the redox couple [Fe(CN)(6)](3)(-)/[Fe(CN)(6)](4)(-). Self-assembled monolayers of mercaptopropane-N-mono-6-deoxy-β-cyclodextrin amide (MPA-CD) on gold surfaces were formed with coverage of 99-100%. The inclusion of charged guest molecules was detected by monitoring the changes in the charge-transfer resistance, which is sensitive to the surface charge density in terms of repulsion or attraction of the redox active ions. Adsorption of positively charged 1-adamantanamino hydrochloride (1-ADHC) led to a considerable increase in the charge-transfer resistance, whereas the inclusion of both negatively charged 1-adamantanecarboxylic acid (1-ADC) and 2-(p-toluidinyl)naphthalene-6-sulfonate (2,6-TNS) caused a decrease. Applying the Frumkin correction to obtain the surface charge density and the Gouy-Chapman-Stern theory to account for the electrochemical double layer, we were able to quantify the binding of the charged guest molecules in terms of binding isotherms. The isotherms display a distinct two step adsorption process probably owing to the presence of two energetically different binding sites on the surface. Complete reversibility of the binding process of the guest molecules could be demonstrated by the addition of β-cyclodextrin in solution, which allowed the reuse of the functionalized surfaces.

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The spontaneous insertion of an anion selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense was monitored by using solid supported lipid membranes (SSMs) composed of the positively charged lipid N,N-dimethyl-N,N-dioctadecylammonium bromide immobilized via electrostatic interaction on a negatively charged monolayer of 3-mercaptopropionic acid chemisorbed on gold. By means of impedance spectroscopy, conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the SSMs. Variation in the dc potential revealed that CAC exhibits its unique voltage dependence in SSMs described by an exponential function in accordance to previous results obtained from patch clamp measurements. Protease treatment and the addition of an anion selective channel inhibitor diminished the increased conductivity of the membrane after CAC addition clearly indicating that the increased conductivity is attributed to the specific insertion of the channel protein. The results demonstrate the potential of this kind of electrostatically attached solid supported membrane combined with impedance spectroscopy allowing one to monitor and characterize water-soluble spontaneously inserting channel forming bacterial toxins.

Solid-supported membranes immobilized on gold electrodes were used to detect and characterize the spontaneously inserting anion-selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense. Three different membrane systems varying in the composition of the first chemisorbed monolayer were investigated by means of impedance spectroscopy. Conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the solid-supported membranes, indicating that the relative change in conductance is largest if the lipid layer is attached to the surface via a flexible lipid anchor. Variation in the d.c. potential revealed that CAC exhibits a voltage dependence in these tethered membranes which can be described by an exponential function in accordance with previous results obtained from patchclamp measurements and impedance analysis. The addition of an inhibitor that selectively blocks anion channels abolished the channel conductance almost completely, indicating that the increased conductivity can be attributed to the specific insertion of the CAC. A linear dependence of the channel conductance on the chloride concentration was found, which was modulated by the charges of the second lipid monolayer. The results demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-soluble spontaneously inserting channels, providing an effective means to probe for bacterial toxins.