P-066

Gerda Žižiūnaitė

gerda.ziziunaite@chgf.vu.lt

Gintaras Valinčius, Aušra Valiūnienė

Faculty of Chemistry and Geosciences, Vilnius University, Lithuania

Kinetics of α-Hemolysin Pore Formation in Tethered Bilayer Membranes Characterized by Fast Fourier transform Electrochemical Impedance Spectroscopy

Tethered bilayer lipid membranes (tBLMs) are structures consisting of a conductive substrate on which a phospholipid bilayer is immobilized via molecular anchors. Such systems mimic biological membranes. tBLMs are used to investigate membrane-related phenomena, such as ion exchange or protein interactions, e.g., pore formation by toxins like mellitin or α-hemolysin. Structural changes in tBLMs caused by pore-forming toxins can be employed creating biological sensors. Similarly, real biological membranes can be damaged by such toxins. The analyte in our work, α-hemolysin, exemplifies this, as its presence in the human body can cause sepsis, pneumonia, etc. Rapid detection is crucial for increasing recovery chances.

Electrochemical impedance spectroscopy (EIS) is a common alternating current method for analyzing tBLMs. Fast Fourier transform allows quick registration of EI spectra, facilitating the investigation of fast kinetic processes. EI spectra provide information on the structural properties of tBLMs, such as capacitances of self-assembly monolayers and phospholipid bilayers. Additionally, EIS spectra can predict system heterogeneity and quantitatively analyze naturally occurring or membrane-damaging species. The limitation of solely using EI spectra for tBLM characterization lies in ambiguous conclusions due to complex system geometry. tBLMs are bathed in bulk solution, but ions in the submembrane reservoir have limited mobility due to geometrical restrictions. Such conditions cannot be accurately reproduced using equivalent schemes with known physical meanings behind each element.

In this work, we created tBLMs using glass slides covered with a fluorine-doped tin oxide (FTO) layer, silanized using a mixture of allyltrichlorosilane (ATS) and trichloro(3-(octadecylthio)propyl)silane (TOPS). The phospholipid bilayer, consisting of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, was immobilized via multilamellar vesicle fusion. We incubated the tBLMs with α-hemolysin and analyzed the EIS data using a mathematical algorithm that characterizes the system as a probability function of defect density (P(Ndef)). From this, we determined the average naturally occurring defect density in tBLMs, which is linearly proportional to the rate of α-hemolysin pore formation. This discovery enables the creation of an α-hemolysin biosensor with a reproducible response, predicted by the qualities of the formed tBLM.