FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices

Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/(B)anti-CD81 and anti-CD63/(B)anti-CD9), resulting in 10(3) and 10(4) times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/(B)anti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCA M antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis

Flexible and Hollow Micro Ring Electrode Arrays for Multi-Directional Monitoring of 3D Neuronal Networks

International audienceThree-dimensional (3D) cell culture based in vitro models shown great promise and able to replicate partially the development and complexity of in vivo nerve tissue. The functional characterization of 3D cell cultures is challenging. Because, most of the in vitro neural interfacing technologies are developed for 2D cell cultures, where it limits the access to the networks within the cultures. Hence, high-performance electrophysiological platforms that allow seamless integration with soft and stable tissue system, and multi-directional bio interfacing capabilities for long term are required.In this context, we report for the first time the fabrication of ‘Stargate’ look alike flexible and hollow micro-ring electrode array (SG MEA) with hole at the centre (40-μm to 150-μm diameter). The SG MEA was microfabricated on a 20-μm thick layer of flexible and biocompatible parylene C configured with 4 to 6 electrode sites. All gold electrode rings were coated with the conducting polymer poly(3,4-ethylenedioxythio-phene):poly(styrene-sulfonate) (PEDOT:PSS) to lower the impedance (5-8 kΩ) and obtain a better signal-to-noise ratio during extracellular recordings. The hollow ring shape of SG electrode architectures, with geometrical surface area of 289 μm2 to 1299 μm2 were designed specifically in such a way that it offers more freedom to the internal dynamics of cell cultures and multidirectional sensing of neuron activities within the 3D neuronal network, from both front and back-end of the SG MEA

Flexible and Hollow Micro Ring Electrode Arrays for Multi-Directional Monitoring of 3D Neuronal Networks

International audienceThree-dimensional (3D) cell culture based in vitro models shown great promise and able to replicate partially the development and complexity of in vivo nerve tissue. The functional characterization of 3D cell cultures is challenging. Because, most of the in vitro neural interfacing technologies are developed for 2D cell cultures, where it limits the access to the networks within the cultures. Hence, high-performance electrophysiological platforms that allow seamless integration with soft and stable tissue system, and multi-directional bio interfacing capabilities for long term are required.In this context, we report for the first time the fabrication of ‘Stargate’ look alike flexible and hollow micro-ring electrode array (SG MEA) with hole at the centre (40-μm to 150-μm diameter). The SG MEA was microfabricated on a 20-μm thick layer of flexible and biocompatible parylene C configured with 4 to 6 electrode sites. All gold electrode rings were coated with the conducting polymer poly(3,4-ethylenedioxythio-phene):poly(styrene-sulfonate) (PEDOT:PSS) to lower the impedance (5-8 kΩ) and obtain a better signal-to-noise ratio during extracellular recordings. The hollow ring shape of SG electrode architectures, with geometrical surface area of 289 μm2 to 1299 μm2 were designed specifically in such a way that it offers more freedom to the internal dynamics of cell cultures and multidirectional sensing of neuron activities within the 3D neuronal network, from both front and back-end of the SG MEA

Carbon Nanofiber/PEDOT Based Macro-Porous Composite for High Performance Multifunctional Neural Microelectrode

International audienceBrain study methods mainly rely on neurochemical detection and electrophysiology, respectively studying the molecular dynamics and electrical activity in the brain cellular environment. The combined and simultaneous use of both study modes holds great promises for patients and researchers, in line with the treatment research but rely on multifunctional platforms (materials, devices, techniques...) still to be developed. Here, we present a new macro-porous composite material made up of PEDOT and carbon nanofibers (CNFs), coated on a flexible neural microelectrode array. A fully controlled one-shot electrodeposition strategy was developed to coat flexible Au-microelectrode surfaces with macro-porous CNF/PEDOT nanocomposite. The oxidized CNFs were used as a dopant and infused within the conductive PEDOT, electrochemically, in such a way that their excellent electronic, mechanical, chemical properties, and resulting combined performances can be exploited to yield better quality neural recordings, electrical stimulation and neurotransmitter detection

Carbon Nanofiber/PEDOT Based Macro-porous Composite For High Performance Multifunctional Neural Microelectrode

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Nanofibrous PEDOT-Carbon Composite on Flexible Probes for Soft Neural Interfacing

International audienceWord count: 199 In this study, we report a flexible implantable 4-channel microelectrode probe coated with highly porous and robust nanocomposite of poly(3,4-ethylenedioxythiophene) (PEDOT) and carbon nanofiber (CNF) as a solid doping template for high-performance in vivo neuronal recording and stimulation. A simple yet well-controlled deposition strategy was developed via in situ electrochemical polymerization technique to create a porous network of PEDOT and CNFs on a flexible 4-channel gold microelectrode probe. Different morphological and electrochemical characterizations showed that they exhibit remarkable and superior electrochemical properties, yielding microelectrodes combining high surface area, low impedance (16.8 ± 2 MΩ.µm2 at 1 kHz) and elevated charge injection capabilities (7.6 ± 1.3 mC/cm²) that exceed those of pure and composite PEDOT layers. In addition, the PEDOT-CNF composite electrode exhibited extended biphasic charge cycle endurance, resulting in a negligible physical delamination or degradation for long periods of electrical stimulation. In vitro testing on mouse brain slices showed that they can record spontaneous oscillatory field potentials as well as single-unit action potentials and allow to safely deliver electrical stimulation for evoking field potentials. The combined superior electrical properties, durability and 3D microstructure topology of the PEDOT-CNF composite electrodes demonstrate outstanding potential for developing future neural surface interfacing applications

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (labs = 572 nm) and yields strong fluorescence (lfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays’mechanism and about their observed discrepancy.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Ring nanoelectrodes integrated into microwell arrays for the analysis of mitochondria isolated from leukemic cells

International audienceWe report here the fabrication and the electrochemical characterization of recessed disk microelectrodes (rDME) and ring nanoelectrodes (rRNE) integrated into microwell arrays. The technological process based on the reactive ion etching of a SiO2/Ti/Pt/Ti/SiO2 stack is optimized in order to realize functional electrochemical microdevices on glass substrate and so, enable the coupling of amperometric measurements with optical analysis. Multiphysic simulations and electrochemical characterizations are carried out to study and enhance the amperometric performance of recessed ring nanoelectrodes arrays (rRNEA) according to their geometry. Finally, all these results demonstrate that rRNEA are fitted for the detection of bio-electrochemical species at the microscale and consequently, for the analysis of the metabolic status of isolated mitochondria through the measurement of dissolved oxygen and hydrogen peroxide

Monitoring metabolic responses of single mitochondria within poly(dimethylsiloxane) wells: study of their endogenous reduced nicotinamide adenine dinucleotide evolution.

It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion

Coupling fluorescence microscopy and electrochemistry to investigate single mitochondria metabolism

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