Amperometry and intracellular vesicle impact electrochemical cytometry with nanotip electrodes were used to monitor the effects on exocytosis and vesicular storage after nano-injection of phospholipids with different geometries into secretory cells.
We present a pH nanosensor
conceived for single intracellular measurements.
The sensing architecture consisted of a two-electrode system evaluated
in the potentiometric mode. We used solid-contact carbon nanopipette
electrodes tailored to produce both the indicator (pH nanosensor)
and reference electrodes. The indicator electrode was a membrane-based
ion-selective electrode containing a receptor for hydrogen ions that
provided a favorable selectivity for intracellular measurements. The
analytical features of the pH nanosensor revealed a Nernstian response
(slope of −59.5 mV/pH unit) with appropriate repeatability
and reproducibility (variation coefficients of <2% for the calibration
parameters), a fast response time (<5 s), adequate medium-term
drift (0.7 mV h
–1
), and a linear range of response
including physiological and abnormal cell pH levels (6.0–8.5).
In addition, the position and configuration of the reference electrode
were investigated in cell-based experiments to provide unbiased pH
measurements, in which both the indicator and reference electrodes
were located inside the same cell, each of them inside two neighboring
cells, or the indicator electrode inside the cell and the reference
electrode outside of (but nearby) the studied cell. Finally, the pH
nanosensor was applied to two cases: (i) the tracing of the pH gradient
from extra-to intracellular media over insertion into a single PC12
cell and (ii) the monitoring of variations in intracellular pH in
response to exogenous administration of pharmaceuticals. It is anticipated
that the developed pH nanosensor, which is a label-free analytical
tool, has high potential to aid in the investigation of pathological
states that manifest in cell pH misregulation, with no restriction
in the type of targeted cells.
We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (∼20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.
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