Traduction technique français anglais de la thèse de doctorat d’Amar TAMRA intitulée “Spectroscopie diélectrique HyperFréquence des cellules biologiques soumises à l’électroporation”
Université Toulouse 3 Paul Sabatier; Unité de recherche : LAAS-CNRS/IPBS
Copyright : Amar TAMRA 2017
French english scientific translation series : Phd thesis entitled : “Microwave dielectric spectroscopy of biological cells under electroporation”.
Phd Student : Amar TAMRA
University : Toulouse 3 Paul Sabatier University
Research Units : The Systems Analysis and Architecture Laboratory (LAAS) & the Institute of Pharmacology and Structural Biology (IPBS) of Toulouse.
Starting with the 1980s, electric fields were being used increasingly in the fields of biology and medicine due to their innovative and promising characteristics offering important therapeutic interests. More particularly, they are increasingly developed and used in the field of cellular electroporation where electrical impulses are applied to cells or tissues in order to induce changes in the properties of the plasma membrane, which becomes temporarily permeable to certain target molecules. This approach lead to the development of new research methods such as electrochemotherapy (transfer of cytotoxic molecules), electrogenotherapy (transfer of DNA or RNA molecules), and irreversible electroporation (ablation of tumors or eradication of microorganisms). Analysis of the effects induced by these phenomena is often carried out using optical or biochemical methods. Optical methods were heavily researched and proved to be powerful tools in biology. Nevertheless, they do have a few disadvantages, such the utilization of a wide range of fluorochromes and markers whose use and setup is both laborious and costly. They are also invasive in nature, given that the cells are altered after being characterized by these methods.
With the development of microelectronics and microtechnologies, we are witnessing a global trend towards the transposition of analysis methods to the micrometric scale, which enables the reduction of the lengths and costs of operations, while integrating several massively parallelizable operations. Besides, a return to the single cell level seems be crucial to the understanding of numerous biological phenomena which would otherwise be hidden by averaging on a cell population. That is why it is interesting to develop new micrometric systems dedicated to the single cell. This technological solution allows us, not only to combine the advantages previously mentioned (reducing the length and cost of operations, parallelization, analysis of individual cells) but also opens up a great opportunity: that of integrating the electrical analysis methods specifically used in biology and which do not require the utilization of specific markers which might alter the cellular response and bias experimental results.
That is why we can find in the literature, innovative micrometric systems designed for the purposes of electrical analysis of cells under electroporation. Most of these works are based on the use of dielectrophoresis (DEP) or low frequency electromagnetic waves (impedance spectroscopy). These tools are in fact very powerful when it comes to revealing the effects induced at the cellular membrane level. But then, what happens beyond the membrane? In order to answer this question, we have to investigate intracellular content, hence the need for ultra-high frequencies (a few GHz). In this frequency range, the membrane becomes transparent to electromagnetic waves, which will then penetrate the cell and interact with the intracellular content. This analysis technique is called microwave dielectric spectroscopy and is proving to be a relevant method that complements existing techniques, as far as the analysis of biological cells is concerned.
In this thesis, which is located at the intersection of three fields (cellular biology, microwave electronics and microtechnology), we present the development of an attractive and competitive analytical method, adapted to the study of the effects of electroporation on individual cells, whose analysis requires neither marking nor contact with the studied cells and which is carried out in the liquid culture medium: microwave dielectric spectroscopy. Our main objective is then to show the usefulness of the dielectric analysis technique in the gigahertz frequency domain for the study of biological cells under electroporation.
Work on this thesis was carried out thanks to the joint logistical support of two teams belonging to two research laboratories representing different scientific fields. The first host team is the Fluidic/Microwave Micro/Nanosystems (MH2F) team of the Systems Analysis and Architecture Laboratory of Toulouse (LAAS-CNRS), within which was undertaken the design and microfabrication of our microdevices, as well as the microwave characterization of various biological samples. The second host team is the Cellular Biophysics team of the Institute of Pharmacology and Structural Biology of Toulouse (IPBS), within which the vital step of characterization and «calibration» of the cells was performed, as well as the development of the protocols corresponding to electroporation.
Our thesis is divided into three main parts:
- The first chapter is a general introduction to the different principles used in the thesis. A detailed explanation of electroporation, lab-on-a-chip, as well as microwave dielectric spectroscopy is provided.
- The second chapter deals with principle of «on-chip» electroporation. To this end, we start with a review of the microsystems dedicated to electroporation at the micrometric scale. This is then followed by the presentation of our microcomponent dedicated to on-chip electroporation as well as the results obtained.
- The third and last chapter is dedicated to the analysis by microwave dielectric spectroscopy of the effects of on-chip electroporation. This chapter introduces the equipment and methods used, the various results of HF spectroscopy for seven distinct biological tests conducted in three experimental setups, as well as the biological « countermeasures » which will correlate with our microwave results. The results obtained throughout the work on this thesis will thus demonstrate that dielectric spectroscopy associated with microfluidics is a reliable and powerful technique, able to enrich our understanding of biological cells under treatment.
Read Next : Chapter 1: Introduction and research objectives