Ultra-sensitive bioelectronic transducers for extracellular electrophysiological studies

Extracellular electrical activity of cells is commonly recorded using microelectrode arrays (MEA) with planar electrodes. MEA technology has been optimized to record signals generated by excitable cells such as neurons. These cells produce spikes referred to as action potentials. However, all cells produce membrane potentials. In contrast to action potentials, electrical signals produced by non-excitable or non-electrogenic cells, do not exhibit spikes, rather smooth potentials that can change over periods of several minutes with amplitudes of only a few microvolts. These bioelectric signals serve functional roles in signalling pathways that control cell proliferation, differentiation and migration. Measuring and understanding these signals is of high priority in developmental biology, regenerative medicine and cancer research. The objective of this thesis is to fabricate and characterise bioelectronic transducers to measure in vitro the bioelectrical activity of non-electrogenic cells. Since these signals are in the order of few microvolts the electrodes must have an unrivaled low detection limit in the order of hundreds of nanovolts. To meet this challenge a methodology to analyze how bioelectrical signals are coupled into sensing surfaces was developed. The methodology relies on a description of the sensing interface by an equivalent circuit. Procedures for circuit parameter extraction are presented. Relation between circuit parameters, material properties and geometrical design was established. This knowledge was used to establish guidelines for device optimization. The methodology was first used to interpret recordings using gold electrodes, later it as extended to conducting polymers surfaces (PEDOT:PSS ) and finally to graphene electrolyte-gated transistors. The results of this thesis have contributed to the advance of the knowledge in bioelectronic transducers in the following aspects: (i) Detection of signals produced by an important class of neural cells, astrocyte and glioma that thus far had remained inaccessible using conventional extracellular electrodes. (ii) Development of an electrophysiological quantitative method for in vitro monitoring of cancer cell migration and cell-to-cell connections. (iii)An understanding of the limitations of electrolyte-gated transistors to record high frequency signals.A atividade elétrica extracelular das células é geralmente medida usando matrizes de micro-elétrodos (MEA) planares. A tecnologia MEA foi otimizada para medir sinais gerados por células excitáveis, como os neurónios. Essas células produzem sinais conhecidos como potenciais de ação. No entanto, todas as células produzem potenciais de membrana. Em contraste com os potenciais de ação, os sinais elétricos gerados por células não excitáveis ou não eletrogénicas, não são “spikes”, mas sinais que variam lentamente e que podem mudar ao longo de períodos de vários minutos com amplitudes de apenas alguns microvolts. Estes sinais desempenham funções importantes nos mecanismos de sinalização que controlam a proliferação, a diferenciação e a migração celular. Medir e entender esses sinais é importante na biologia do desenvolvimento, na medicina regenerativa e no desenvolvimento de novas terapias para combater células cancerosas. O objetivo desta tese é fabricar e caracterizar transdutores para medir in vitro a atividade de células não eletrogénicas. Como esses sinais são da ordem de alguns microvolts, os elétrodos devem ter um limite de detecção na ordem de centenas de nanovolts. Para enfrentar este desafio, foi desenvolvida uma metodologia para analisar a forma como os sinais se acoplam à superfície do sensor. A metodologia baseia-se na descrição da interface de detecção por um circuito eléctrico equivalente. Procedimentos para extração dos parâmetros de circuito e a relação com as propriedades do material e o desenho geométrico foi estabelecida. Este conhecimento foi usado para estabelecer diretrizes para otimização dos transdutores. Em primeiro lugar a metodologia foi usada para interpretar as medidas de sinais usando elétrodos de ouro, posteriormente estendida para analisar superfícies de polímeros condutores (PEDOT: PSS) e, finalmente, para compreender o funcionamento de transístores. Os resultados desta tese contribuíram para o avanço do conhecimento em transdutores bioeletrónicos nos seguintes aspectos: (i) Detecção de sinais produzidos por uma importante classe de células neurais, astrócitos e gliomas, que tem permanecido inacessíveis usando elétrodos extracelulares. (ii) Desenvolvimento de um método eletrofisiológico para medir a migração de células cancerosas e o estabelecimento de conexões entre células. (ii) Estudo das limitações dos transístores para medir sinais eletrofisiológicos rápidos.The work developed in this thesis was carried out within the framework of the project entitled: “Implantable Organic Devices for Advanced Therapies (INNOVATE)”, ref. PTDC/EEI-AUT/5442/2014, financed by Fundação para a Ciência e Tecnologia (FCT).This project was carried out at the laboratories of the “ Instituto de Telecomunicações (IT) UID/Multi/04326/2013” at the University of the Algarve. The PhD study period received full scholarship under European EM program, “Erasmus Mundus Action 2 (EMA2)” coordinated by University of Warsaw

Extracellular electrophysiological measurements of cooperative signals in astrocytes populations

Astrocytes are neuroglial cells that exhibit functional electrical properties sensitive to neuronal activity and capable of modulating neurotransmission. Thus, electrophysiological recordings of astroglial activity are very attractive to study the dynamics of glial signaling. This contribution reports on the use of ultra-sensitive planar electrodes combined with low noise and low frequency amplifiers that enable the detection of extracellular signals produced by primary cultures of astrocytes isolated from mouse cerebral cortex. Recorded activity is characterized by spontaneous bursts comprised of discrete signals with pronounced changes on the signal rate and amplitude. Weak and sporadic signals become synchronized and evolve with time to higher amplitude signals with a quasi-periodic behavior, revealing a cooperative signaling process. The methodology presented herewith enables the study of ionic fluctuations of population of cells, complementing the single cells observation by calcium imaging as well as by patch-clamp techniques.Portuguese Foundation for Science and Technology (FCT) [PTDC/EEI-AUT/5442/2014]; Instituto de Telecomunicacoes [UID/Multi/04326/2013]; Associated Laboratory - Institute of Nanoscience and Nanotechnology [POCI-01-0145-FEDER-016623]; [PTDC/CTM-NAN/3146/2014]info:eu-repo/semantics/publishedVersio

Bioelectrical signal detection using conducting polymer electrodes and the displacement current method

Conducting polymer electrodes based on poly (3, 4 ethylenedioxythiophene): polystyrene sulfonate were used to record electrophysiological signals from autonomous cardiac contractile cells present in embryoid bodies. Signal detection was carried out by measuring the displacement current across the polymer/electrolyte double-layer capacitance, and compared with voltage detection. While for relatively low capacitance electrodes, the voltage amplification provides higher signal quality, and for high capacitive electrodes, the displacement current method exhibits a higher signal-to-noise ratio. It is proposed that the displacement current method combined with high capacitive polymer-based electrodes is adequate to measure clusters of cells and whole organs. Our approach has a great potential in fundamental studies of drug discovery and safety pharmacology.Portuguese Foundation for Science and Technology through the Implantable Organic Devices for Advanced Therapies Project [PTDC/EEI-AUT/5442/2014]Instituto de Telecomunicacoes [UID/Multi/04326/2013]info:eu-repo/semantics/publishedVersio

Extracellular electrophysiological based sensor to monitor cancer cells cooperative migration and cell-cell connections

Herein, we describe an electrophysiological based sensor that reproducibly monitors and quantifies in real-time collective migration and the formation of cell-cell junctions by C6 glioma cells seeded on top of electrodes. The signal amplitude and frequency generated by the migrating cells changed over time and these parameters were used to accurately calculate the migration speed. Electrophysiological measurements could also distinguish individual from collective cell migration. The migration of densely packed cells generated strong signals, while dispersed cells showed weak bioelectrical activity. We propose this electrophysiological technique as a cell-based biosensor to gain insight into the mechanisms of cooperative migration of cancer cells. Possible applications include screening for anti-migratory compounds, which may lead to the development of novel strategies for antineoplastic chemotherapy.Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [PTDC/EEI-AUT/5442/2014, UID/EEA/50008/2019, UID/BIM/04773/2019, UID/Multi/04326/2019]Universidade do Algarv