Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry (original) (raw)
Related papers
In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 μm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.
2011
A novel nanocapillary electrophoretic electrochemical (Nano-CEEC) chip has been developed to demonstrate the possibility of zeptomole-level detection of neurotransmitters released from single living cells. The chip integrates three subunits to collect and concentrate scarce neurotransmitters released from single PC-12 cells, including a pair of targeting electrodes for single cells captured by controlling the surface charge density; a dual-asymmetry electrokinetic flow device for sample collection, pre-concentration and separation in a nanochannel; and an online electrochemical detector for zeptomole-level sample detection. This Nano-CEEC chip integrates a polydimethylsiloxane microchannel for cell sampling and biomolecule separation and a silicon dioxide nanochannel for sample pre-concentration and amperometric detection. The cell-capture voltage ranges from 0.1 to 1.5 V with a frequency of 1-10 kHz for PC-12 cells, and the single cell-capture efficiency is optimized by varying the duration of the applied field. All of the processes, from cell sampling to neurotransmitter detection, can be completed within 15 min. Catecholamines, including dopamine and norepinephrine (noradrenaline) released from coupled single cells, have been successfully detected using the Nano-CEEC chip. A detection limit of 30-75 zeptomoles was achieved, which is close to the levels released by a single neuron in vitro.
Biosensors and Bioelectronics, 2012
Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 m × 300 m) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 M, with a sensitivity of 23.1 ± 0.5 mA/M mm 2 . The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50 mM) containing Tyrode's solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ∼52 M. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.
Vesicular exocytosis and microdevices – microelectrode arrays
The Analyst, 2015
Among all the analytical techniques capable of monitoring exocytosis in real time at the single cell level, electrochemistry (particularly amperometry at a constant potential) using ultramicroelectrodes has been demonstrated to be an important and convenient tool for more than two decades.
Biosensors and Bioelectronics, 2009
Size-controllable micron or nano-disk carbon fiber electrode (CFE) is prepared and demonstrated to be excellent for extra-cellular transmitter release detection at tiny structures and vesicle fusion kinetics analysis with high spatio-temporal resolution. An improved electrochemical etching procedure was employed, for the first time, to fabricate cylindrical fiber with controlled micron or nano-diameter. Afterwards, a facile insulation with polypropylene sheath was employed to completely insulate the whole body of the thinned fiber, and an ultrasmall-disk sensing area was finally produced by cutting of the insulated fibers. Scanning electron microscopy (SEM) was employed to characterize the ultrasmall geometry size of the fabricated electrode and to show the tight adherence of the insulation sheath on the fiber. The cut ends of the electrodes were also shown to be smooth, clean and without obvious jagged layer. The fabricated micron or nano-disk carbon electrodes show ideal steady-state voltammetric behavior with satisfying reversibility. Subsequently, the performance of the ultrasmall-disk CFE for amperometric detection of cell secretion was characterized. Results showed that, compared to the conventional micro-disk CFE, the etched small disk CFE possesses higher sensitivity due to its obviously improved signal-to-noise level, which enables minute amounts of 3000 oxidizable molecules to be detectable. The nano-disk CFE was shown to be particularly ideal for analysis of fusion kinetics, due to its avoidance of diffusion broadening of the detected spikes, which is the inherent defect of the conventional micro-CFE technique.
Scientific Reports, 2013
Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the ''weak base hypothesis'' that amphetamine reduces synaptic vesicle transmitter and quantal size.
Microelectrodes for the Measurement of Catecholamines in Biological Systems
Analytical Chemistry, 1996
Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-µm diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that highpass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo. Many chemical messengers that relay information between biological cells can be electrochemically oxidized. These molecules include catecholamines, 1-3 histamine, 4 5-hydroxytryptamine, 5-7 and insulin. 8 For this reason, microsensors designed from carbon fibers have been shown to be quite useful to monitor secretion of these species in a variety of biological systems. 1-8 As progress is made in these measurements, it is clear that the optimum type of electrode and electroanalytical technique depend upon the particular biological preparation investigated. Four major issues in electrochemical measurements are electrode response time and electrode size, as well as sensitivity and selectivity of the method. These issues are often intertwined. For example, early studies showed that differential pulse voltammetry at electrochemically modified electrodes had high selectivity but could not resolve the rapid concentration changes that neurotransmitters undergo. 9-12 This was due to the slow response time of the electrodes as well as the slow scan rate of the technique. The two techniques most widely used today are amperometry 3,14-18 and fast-scan cyclic voltammetry. 5,19-22 Amperometry allows measurement of the most rapid concentration changes and is theoretically limited by the double-layer capacitance of the electrode and the resistance of the surrounding solution, but it provides no chemical identification of the species detected. 3,14-16 Nevertheless, amperometry has provided unique information on the kinetics of secretion and subsequent biochemical fates of the easily oxidized chemical messengers in situations where the biological environment is well controlled. Such situations arise in cell cultures containing a single cell type, 1,18,23,24 or in the brain when specific nerve terminals are activated. 3,14 Fastscan cyclic voltammetry is particularly useful because the voltammogram provides information to identify the detected substance. 25,26 However, cyclic voltammetry's response time, selectivity, and sensitivity critically depend on the electrode's surface state. 6,27
2021
We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption-release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between chemical cargo and vesicle ...
Proceedings of the National Academy of Sciences of the United States of America, 1991
Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.