Table 1.
Refractive indices of the applied solutions determined by OWLS assays in transverse magnetic (ncTM) mode.
Figure 1.
OWLS recordings of deposition of multiple liposome layers and permeability for small organic cations.
(A) Biotinylated sensors were treated with (1) NeutrAvidin; (2) biotin-ssDNA; (3) Chol-dsDNA1-tagged liposomes; (4) Chol-dsDNA2. The insert shows the increasing thickness (dA) of the deposited material on the surface reaching the detectable maximum (∼200 nm) after the second injection of liposomes. (B, C) Changes of the effective refractive indices (NTM) in a representative series of experiments with cross-linked multilayer of liposomes without (B) or with (C) gramicidin channels after injections of ethanolamine (5), methylamine (6) or guanidine (7) solutions.
Figure 2.
Schematic view of the membrane-sandwich sensor set-up.
A thick (140 µm) PTFE lipid-holder membrane was placed on the top of a thin (23 µm) PET membrane for complete separation of lipid material from the sensor surface.
Figure 3.
Sensitivity of different sensor set-ups to selected ions.
Relative NTM values were determined as functions of ethanolamine (A), guanidine (B) and Cl− (C, D) concentrations using different sensor set-ups including bare sensors and sensors furnished with empty PET+PTFE membranes, PET+PTFE membranes+liposomes, PET+PTFE membranes filled with cell-derived biomembranes and PET+PTFE membranes filled with cell-derived biomembranes+liposomes. Relative NTM (%) were calculated as percentages of NTM values detected at the highest (150 mM) compound concentration (100%) for each sensor set-up. The inserts show representative series of data in semi-logarithmic plots for give a higher resolution at low concentrations.
Table 2.
The minimum amount of detectable material in assays with various sensor set-ups.
Figure 4.
Detection of cation migration through gramicidin channels in membrane-sandwich sensor set-ups.
Refractive indices (ncTM) of the sensor-covering fluid layer were measured in the transverse magnetic (TM) mode in sensor set-ups containing empty or lipid-filled filter membranes (A, B) after injecting ethanolamine (A) or guanidine (B) solutions, and after the incorporation of gramicidin (C, D). The inserts show averages and standard deviations of ncTM changes, calculated from three independent experiments. Significant (***p<0.001; paired t-test) differences were found in response to filling with liposomes regardless of the chemical nature of the analyte (A, B). Incorporation of gramicidin, while resulted in enhanced permeation of ethanolamine (C), did not cause detectable changes in the move of guanidine (D).
Figure 5.
Demonstration of Cl–-channel functions of cell-derived GABA receptors in the membrane-sandwich sensor set-up.
OWLS recordings were made in Cl–-free ACSF as running buffer with injection of Cl–-containing ACSF (ACSF) with or without GABA and the channel blocker bicuculline (ACSF+GABA and ACSF+GABA+Bic, respectively). (A) Changes of the effective refractive index (NTM) values in a representative OWLS assay. (B) Changes in the refractive index (ncTM) in response to the GABA-channel blocker bicuculline are shown from a representative experiment. (C) Summary of refractive index (ncTM) changes (ΔncTM) in response to transfusion with Cl–-containing buffer (ACSF), in the presence of the agonist GABA (ACSF+GABA) or in the presence of both GABA and the channel-blocker bicucullin (ACSF+GABA+bicucullin). ΔncTM values were calculated from data of 4 independent series of experiments (n≥4); averages and standard deviations are presented.