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

Transmembrane potential induced on the internal organelle by a time-varying magnetic field: a model study

<p>Abstract</p> <p>Background</p> <p>When a cell is exposed to a time-varying magnetic field, this leads to an induced voltage on the cytoplasmic membrane, as well as on the membranes of the internal organelles, such as mitochondria. These potential changes in the organelles could have a significant impact on their functionality. However, a quantitative analysis on the magnetically-induced membrane potential on the internal organelles has not been performed.</p> <p>Methods</p> <p>Using a two-shell model, we provided the first analytical solution for the transmembrane potential in the organelle membrane induced by a time-varying magnetic field. We then analyzed factors that impact on the polarization of the organelle, including the frequency of the magnetic field, the presence of the outer cytoplasmic membrane, and electrical and geometrical parameters of the cytoplasmic membrane and the organelle membrane.</p> <p>Results</p> <p>The amount of polarization in the organelle was less than its counterpart in the cytoplasmic membrane. This was largely due to the presence of the cell membrane, which "shielded" the internal organelle from excessive polarization by the field. Organelle polarization was largely dependent on the frequency of the magnetic field, and its polarization was not significant under the low frequency band used for transcranial magnetic stimulation (TMS). Both the properties of the cytoplasmic and the organelle membranes affect the polarization of the internal organelle in a frequency-dependent manner.</p> <p>Conclusions</p> <p>The work provided a theoretical framework and insights into factors affecting mitochondrial function under time-varying magnetic stimulation, and provided evidence that TMS does not affect normal mitochondrial functionality by altering its membrane potential.</p

Electromagnetic probes of molecular motors in the electron transport chains of mitochondria and chloroplasts

We report on measurements of harmonics generated by whole cells, mitochondria, and chloroplasts in response to applied sinusoidal electric fields. The frequency- and amplitude-dependence of the induced harmonics exhibit features that correlate with physiological processes. Budding yeast (S. cerevisiae) cells produce numerous harmonics, the amplitudes of which depend strongly on frequency. When the second or third harmonic amplitude is plotted vs. applied frequency, we observe two peaks, around 3 kHz and 12 kHz, which are suppressed by respiratory inhibitors. We observe similar peaks when measuring the harmonic response of B. indicas, a relative of the mitochondrial ancestor. In uncoupled mitochondria, in which most of the electron transport chain is active but the ATP-synthase molecular turbine is inactive, only one (lower frequency) of the two peaks is present. Finally, we find that harmonics generated by chloroplasts depend dramatically on incident light, and vanish in the absence of light

Investigations of ring nanoelectrodes integrated into microwell arrays for the analysis of isolated mitochondria at the microscale

International audienceMitochondria are known to be the major cellular source for ATP (through the oxidative phosphorylation pathway) but also to play main roles into cell apoptosis and related disorders (ageing, neurological diseases, cancers,...). Consequently, new methodological approaches are resuired in order to decipher mitochondria metabolisms. In this context, we are investigating the ElecWell (electrochemical microwell) technological platform based on the integration of ring nanoelectrodes into microwell arrays. Similar to ultra-microelectrodes, they exhibit high current density, fast response time, reduced charging current and high signal to noise ratio. In addition, the use of small analysis volumes (< 1 pL) makes them well suited for the detection of exocytotic events or for the analysis of single mitochondrion. Consequently, platinum ring nanoelectrodes (RNE) (surface: 10-15 µm 2) were integrated into SiO2-based microwells (radius: 3 and 4.5 µm, depth: 5.2 µm, total volume: < 1 pL, figures 1 and 2). These electrochemical devices were characterized by cyclic voltammetry in Fc(MeOH) solutions using single well or arrayed configurations, and optimised according to COMSOL™ simulations. Steady-state sigmoidal responses were shown for RNE devices in agreement with theoretical models and design laws were defined for RNE arrays in the frame of mitochondria statistical analysis. Finally, the ElecWell platform was tested for the mitochondrial analysis (figure 3), allowing the electrochemical monitoring of oxygen consumption rate in response of specific drugs (ethanol, ADP, antimycine A) for several thousands of mitochondria (figure 4). Fig. 1: Integration of platinum ring nanoelectrodes Fig 2: Fabrication of RNE-based devices into a SiO2-based microwell (radius: 3 m) onto glass substrates Fig. 3: Fluorescent analysis of mitochondria Fig. 4: amperometric monitoring of mitochondrial deposited on a RNE-based microwell array metabolisms during an EtOH/ADP/AA cycl

Investigations of ring nanoelectrodes integrated into microwell arrays for the analysis of isolated mitochondria at the microscale

International audienceMitochondria are known to be the major cellular source for ATP (through the oxidative phosphorylation pathway) but also to play main roles into cell apoptosis and related disorders (ageing, neurological diseases, cancers,...). Consequently, new methodological approaches are resuired in order to decipher mitochondria metabolisms. In this context, we are investigating the ElecWell (electrochemical microwell) technological platform based on the integration of ring nanoelectrodes into microwell arrays. Similar to ultra-microelectrodes, they exhibit high current density, fast response time, reduced charging current and high signal to noise ratio. In addition, the use of small analysis volumes (< 1 pL) makes them well suited for the detection of exocytotic events or for the analysis of single mitochondrion. Consequently, platinum ring nanoelectrodes (RNE) (surface: 10-15 µm 2) were integrated into SiO2-based microwells (radius: 3 and 4.5 µm, depth: 5.2 µm, total volume: < 1 pL, figures 1 and 2). These electrochemical devices were characterized by cyclic voltammetry in Fc(MeOH) solutions using single well or arrayed configurations, and optimised according to COMSOL™ simulations. Steady-state sigmoidal responses were shown for RNE devices in agreement with theoretical models and design laws were defined for RNE arrays in the frame of mitochondria statistical analysis. Finally, the ElecWell platform was tested for the mitochondrial analysis (figure 3), allowing the electrochemical monitoring of oxygen consumption rate in response of specific drugs (ethanol, ADP, antimycine A) for several thousands of mitochondria (figure 4). Fig. 1: Integration of platinum ring nanoelectrodes Fig 2: Fabrication of RNE-based devices into a SiO2-based microwell (radius: 3 m) onto glass substrates Fig. 3: Fluorescent analysis of mitochondria Fig. 4: amperometric monitoring of mitochondrial deposited on a RNE-based microwell array metabolisms during an EtOH/ADP/AA cycl

Microwell Array Integrating Ring Nanoelectrodes for The Monitoring of Metabolic Responses at Isolated Mitochondria

International audienceMitochondria are major cell organelles since they are the main source of ATP owing to the oxidative phosphorylation pathway. They also play an important role into several other metabolic pathways (Krebs cycle, lipid synthesis…) and when defective, they are involved into severe pathologies (myopathies, neurological diseases, cancers...). Consequently, new methodological approaches [1, 2] are required in order to decipher mitochondria metabolisms and to provide efficient tools for diagnosis. In this context, we have developed microsystems, namely ElecWell platforms, which combine electrochemical [1] and optical [2] sensing abilities. These are based on the integration of platinum ring nanoelectrodes (RNE, surface: 10-15 µm 2) into SiO2-based microwell arrays (well radius: 3 to 4.5 µm, depth: 5 µm, individual volume < 1 pL), as shown on figure 1. Similarly to ultramicroelectrodes, RNE exhibit high current density, fast response time, reduced charging current and high signal-to-noise ratio. These electrochemical devices were characterized by cyclic voltammetry using single well or array configurations, and optimised according to COMSOL™ simulations. In addition, the glass substrate of the microsystems allows the observation by microscopy of the content and reactivity within each well. Then, a suspension of isolated mitochondria (yeast origin) was deposited on the ElecWell array and allowed to sediment within wells. We monitored by fluorescence the presence of individual mitochondria within wells (Fig. 1b) owing to their NADH content and variations [2]. Simultaneously, we monitored by cyclic voltammetry the variations of their oxygen consumption rate in response to specific activators and inhibitors of respiratory chain activity (ethanol, ADP, antimycine A). The resolution offered by the ElecWell platform is nearly a few thousands of mitochondria (Fig. 1c), corresponding to the mitochondrial content of a single cell. a) b) c) Figure 1. a) Integration of a platinum ring nanoelectrode into a SiO2-based microwell (radius: 3 µm) within the array (10 6 wells); b) Monitoring by fluorescence (NADH content) of isolated mitochondria deposited within wells. c) Detection by cyclic voltammetry (reduction current sampling) of the oxygen variations during mitochondrial activation (EtOH/ADP) and inhibition (Antimycin A)

Microsystems for the electrochemical and optical monitoring of bioenergetic activities of isolated mitochondria

International audienceMitochondria are known as central players in many cellular processes including oxidative phosphorylation, oxidative stress and signaling through the production of reactive oxygen species, or the activation of apoptosis by the cytochrome c release. Consequently, they play a key role in the progression of diseases linked to ageing, including cancers and neurodegenerative troubles. Thus, lots of efforts are currently devoted to develop innovative therapies based on the modulation of mitochondrial activity. This implies increasing demand for devices allowing the analysis of metabolic processes at the scale of isolated mitochondria. In this context, we developed the ElecWell (electrochemical microwell), based on the integration of ring nanoelectrodes (RNE) into silica microwell arrays made on glass substrates [1]. The new generation of ElecWell devices was adapted to a temperature-controlled microscopy platform. Two planar electrodes were integrated to obtain a complete electrochemical cell and allow experiments in closed-flow-through configuration (Figure 1A). Methods were developed to enhance the filling rate of microwells by mitochondria and to reduce biofouling (Figure 1B). First results were obtained with mitochondria isolated from rodent cardiomyocytes. Oxygen consumption was measured locally by cyclic voltammetry at the RNEs (Figure 1C) whereas individual variations of mitochondrial membrane potential were monitored by fluorescence microscopy (Figure 1D), [2]. Next steps consist in performing simultaneous measurements and to reach the electrochemical detection of the bioenergetic activity of single mitochondria with individually addressable microwells. [1] Sékli Belaïdi F. et al., Sensors Actuators B-Chemical, 2016, 232, 345 [2] Vajrala V.S. et al, Integrative Biology, 2016, 8, 836. Figure 1: (A) the 2 nd generation of the ElecWell device mounted on its microscopy platform; (B) mitochondria into microwells (TMRM fluorescence); (C) variations of dissolved oxygen concentration versus time; (D) variations of mitochondrial membrane potential versus time, both as function of activator/inhibitor additions