Razvoj avtonomnega sistema za merjenje električnih lastnosti ravninskih lipidnih dvoslojev

MONTANI, JAŠA (author), Kramar, Peter (mentor)

Celična membrana in membrane celičnih organelov so v večini sestavljene iz lipidnih dvoslojev, ki vsebujejo tudi proteinske molekule. Izpostavitev celice električnemu polju ustrezne jakosti in trajanja povzroči povečano prepustnost celične membrane. Ta pojav imenujemo elektroporacija. Elektroporacijo danes rutinsko uporabljamo v biomedicini in biotehnologiji. Opravljene raziskave nakazujejo, da se spremembe v membrani – pore zaradi vsiljenega električnega polja pojavijo v lipidnih predelih celične membrane. Kot dober približek celične membrane pri raziskavah na področju elektroporacije velikokrat uporabljamo umetno ustvarjeni ravninski lipidni dvosloj. Ravninski lipidni dvosloj z makroskopskega vidika opišemo z lastnostmi, kot so: kapacitivnost, debelina, upornost oziroma prevodnost in porušitvena napetost. Vsaka izmed teh lastnosti potrebuje svoj princip merjenja. Meritve omenjenih lastnosti večinoma temeljijo na tokovnem oziroma napetostnem vzbujanju ravninskega lipidnega dvosloja in merjenju njegovega napetostnega oziroma tokovnega odziva. Postopki priprave ravninskega lipidnega dvosloja so časovno in tehnično zahtevni. Po postavitvi ravninskega lipidnega dvosloja preverimo ali smo ravninski lipidni dvosloj zgradili z merjenjem toka ob vzbujanju s 100 µs napetostnim pulzom amplitude 100 mV. Če je tok zanemarljivo majhen, izmerimo kapacitivnost po metodi razelektritve pulza ter ocenimo kvaliteto zgrajenega lipidnega dvosloja. Za tem ravninski lipidni dvosloj lahko preučujemo na različne načine. Merimo lahko denimo njegovo upornost in kapacitivnost skozi čas z impedančno metodo ali pa uporabimo linearno naraščajoč signal, da ravninski lipidni dvosloj porušimo in izmerimo porušitveno napetost. Poizkusi so lahko zelo zamudni, saj trajajo tudi po več ur ali cel dan. Namen magistrske naloge je razvoj avtonomnega merilnega sistema, ki poenostavlja, avtomatizira postopke gradnje ravninskega lipidnega dvosloja in merjenja električnih lastnosti. Ciljni uporabnik našega sistema je raziskovalec v laboratoriju. Sistem temelji na prirejenem vezju razvitem v diplomskem delu Roka Maliča. Dodali smo funkcionalnost za avtomatsko tvorjenje lipidnega dvosloja s pomočjo laboratorijskih injekcijskih črpalk WPI ALADDIN-1000, ki jih krmilimo preko protokola UART. Prav tako smo standardizirali tvorjenje vzbujalnih napetostnih signalov in omogočili merjenje toka in napetosti ter analiziranje izmerjenih podatkov. Razvili smo način, da uporabnik s sistemom upravlja preko osebnega računalnika in orodja MATLAB, za katerega smo izdelali enostaven uporabniški vmesnik na podlagi standardnih ukazov SCPI (ang: Standard Commands for Programmable Instruments).

• Language: Slovenian
• Keywords: ravninski lipidni dvosloj, meritve, Red Pitaya, SCPI
• Work type: Master's thesis/paper
• Organization: FE - Faculty of Electrical Engineering
• Year: 2020
• PID: 20.500.12556/RUL-115060
• Publication date in RUL: 10.04.2020

Secondary language

• Language: English
• Title: Development of an autonomous system for measurement of planar lipid bilayer electrical properties

Abstract

The cell membrane and the membranes of cellular organelles are mostly composed of lipid bilayers that also include protein molecules. Exposing a cell to an electrical field of sufficient strength and duration increases the permeability of the cell membrane. This phenomenon is called electroporation. Electroporation is routinely used today in biomedicine and biotechnology. Experiments show that during electroporation the changes in the membrane – pores occur within the lipid regions of the cell membrane. Artificially created planar lipid bilayers are often used as an approximation of the actual cell membrane in electroporation research. Planar lipid bilayers are macroscopically defined by their characteristics such as capacitance, thickness, resistance or conductance, and breaking voltage. Each one of these characteristics requires a specific measurement technique. Measurements are mostly based on voltage or current clamping and then analysing the current or voltage responses respectively. Constructing planar lipid bilayers is time-consuming and technically challenging. After the planar lipid bilayer is constructed we verify its existence by measuring the current when exposing the potential bilayer to a brief 100 µs voltage pulse with an amplitude of 100 mV. If the current is negligibly small, we measure the capacitance with the pulse discharge method and assess the quality of the planar lipid bilayer. Then we can study the planar lipid bilayer in various ways like measuring its resistivity and capacitance in relation to time with impedance-based methods or we can use a voltage signal that increases linearly with time to determine the breaking time and breaking voltage of the planar lipid bilayer. Experiments are time-consuming and can last multiple hours or even span multiple days. The purpose of this study is to develop an autonomous measuring system that simplifies and automates the procedures of creating planar lipid bilayers and measuring their electrical properties. The end-user of our system is a researcher in a laboratory. The system is based on a circuit developed by Rok Malič in his bachelor diploma. We added the functionality of automatic planar lipid bilayer construction with the help of two laboratory syringe pumps WPI ALADDIN-1000, which are controlled with UART communication. Furthermore, we standardized the generation of voltage signals for voltage clamping and enabled measuring electrical current and voltage and further analysis of collected data. We developed a way for the user to control and take advantage of the system with a personal computer and MATLAB, for which we developed a simple user interface following SCPI (Standard Commands for Programmable Instruments) standards.

• Keywords: Planar lipid bilayer, measurement, Red Pitaya, SCPI