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- Bioelectrical signal detection using conducting polymer electrodes and the displacement current methodPublication . Inácio, Pedro; Mestre, Ana L G; Medeiros, C.R.; Asgarifar, Sanaz; ELAMINE, Youssef; Canudo, Joana; Santos, João; Bragança, José; Morgado, Jorge; Biscarini, Fabio; Gomes, Henrique L.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.
- Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populationsPublication . Mestre, Ana L. G.; Cerquido, Monica; INÁCIO, PEDRO; Asgarifar, Sanaz; Lourenco, Ana S.; Lurdes S. Cristiano, M.; Aguiar, Paulo; Medeiros, Maria C. R.; Araújo, Inês; Ventura, Joao; Gomes, Henrique L.Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 mu V for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm(2)) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 mu V in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.
- Extracellular electrophysiological based sensor to monitor cancer cells cooperative migration and cell-cell connectionsPublication . Asgarifar, Sanaz; Mestre, Ana L. G.; Félix, Rute; Inacio, Pedro M. C.; Lurdes S. Cristiano, M.; Medeiros, Maria C. R.; Araújo, Inês; Power, Deborah; Gomes, Henrique L.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.
- Ultra-sensitive bioelectronic transducers for extracellular electrophysiological studiesPublication . Asgarifar, Sanaz; Gomes, Henrique L.; Ruano, M. GraçaExtracellular 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.
- Extracellular electrophysiological measurements of cooperative signals in astrocytes populationsPublication . Mestre, Ana L. G.; INÁCIO, PEDRO; ELAMINE, Youssef; Asgarifar, Sanaz; Lourenco, Ana S.; Cristiano, Maria L. S.; Aguiar, Paulo; Medeiros, Maria C. R.; Araújo, Inês; Ventura, Joao; Gomes, Henrique L.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.