| Abstract [eng] |
Tethered bilayer membranes (tBLM) are used as models that mimic biological cell membranes. In this work, such a model was developed using a glass slide coated with fluorine-doped tin oxide (FTO), on which a phospholipid bilayer (60 % DOPC and 40 % Chol.) was immobilised via self-assembled monolayer (TOPS:ATS). The kinetics of the formation of the phospholipid bilayer and the dynamics of the incorporation of the pore-forming toxin, α-hemolysin, into tBLM were investigated using FFT-EIS. FFT-EIS is unique in that it allows to measure electrochemical impedance spectra very rapidly, within ∼15 s (measuring spectra in the 0.2-10 kHz AC range). In the case of the system studied, the phospholipid bilayer was formed within ∼30 min, independently of the different properties of the tBLM, but kinetic studies showed that the initial kinetics of membrane formation depends on the heterogeneity of the SAM. Also, the trajectory of the electrochemical impedance spectra suggested that the formation of the phospholipid bilayer on the FTO|SAM occurs via the formation of a small number of phospholipid bilayer “islands“, which expand as the membrane forms. Changes in the electrochemical impedance spectra induced by αHL damage are also observed already within the first minute after the onset of αHL exposure. The detection of αHL aids in the diagnosis of infections, including skin and soft tissue infections, pneumonia and sepsis - early detection of the infection can help prevent potentially fatal complications. Traditional methods for αHL detection, such as culturing bacteria, are not suitable for rapid detection of αHL. For this reason, the ultra-fast detection of αHL toxin by FFT-EIS would be very attractive for the development of an αHL biosensor. In this work, tBLM was incubated using the same, 100 nM concentration of αHL to investigate how the properties of tBLM influence the kinetics and extent of αHL exposure. The work revealed that the kinetics of αHL exposure varies between different tBLMs, which is likely to be strongly influenced by the heterogeneity of the substrate and the tBLM system itself. Using FFT-EIS, the tendency of αHL to form clusters was also assessed: both the variation of the phase minimum with logarithm (argZmin(logfmin)) observed in electrochemical impedance Bode coordinates and the values of the ζ parameter used to assess the heterogeneity of the system led to the conclusion that αHL in this system tends to form clusters. The different levels of clustering observed are again due to the heterogeneity of the substrate and the tBLM itself. An increase in the rate of clustering with time was also observed in the study of the variation of the ζ parameter with time for some tBLM systems. The EIS method, which is not usually used for structural analysis at the nanometre level, can be used as a tool to estimate the clustering rate of natural and protein-formed defects in tBLM. |
