Carbon fiber ultramicrodic electrode electrodeposited with over-oxidized polypyrrole for amperometric detection of vesicular exocytosis from pheochromocytoma cell

Abstract

Vesicular exocytosis is ubiquitous, but it is difficult to detect within the cells' communication mechanism. For this purpose, a 2 µm ultramicrodic carbon fiber electrode was fabricated in this work based on electrodeposition with over-oxidized polypyrrole nanoparticle (PPyox-CFE), which was applied successfully for real-time monitoring of quantal exocytosis from individual pheochromocytoma (PC12) cells. PPyox-CFE was evaluated by dopamine (DA) solutions through cyclic voltammetry and amperometry electrochemical methods, and results revealed that PPyox-CFE improved the detection limit of DA. In particular, the sensitivity of DA was improved to 24.55 µA·µM(-1)·µm(-2) using the PPyox-CFE. The ultramicrodic electrode combined with the patch-clamp system was used to detect vesicular exocytosis of DA from individual PC12 cells with 60 mM K+ stimulation. A total of 287 spikes released from 7 PC12 cells were statistically analyzed. The current amplitude (Imax) and the released charge (Q) of the amperometric spikes from the DA release by a stimulated PC12 cell is 45.1 ± 12.5 pA and 0.18 ± 0.04 pC, respectively. Furthermore, on average ~562,000 molecules were released in each vesicular exocytosis. PPyox-CFE, with its capability of detecting vesicular exocytosis, has potential application in neuron communication research.

Figure 1.

(A) The fabricated CFE with diameter of 2 μm; the inset shows interface between carbon fiber and the glass insulator; (B) The SEM at the cycle of 8.

Figure 2.

Signal-to-noise (S/N) statistics of current response to 2 μM DA.

Figure 3.

Cyclic voltammetry response of bare CFE was compared to those CFE modified with PPyox the electrochemical in 2 μM DA solution.

Figure 4.

(A) Current-time response of the CFE electrodeposited with PPyox film at different concentrations at 149 mV vs. Ag|AgCl; (B) Detection limit of DA using PPyox-CFE; (C) Plot of current versus concentration of DA in the range of 0.5 μM to 12 μM.

Figure 5.

(A) The traces are recorded by amperometric method continuously for 25 s at the distance of 1 μm (i), 500 μm (ii) from cell with 60 mM K⁺ stimulation solution, respectively; (B) The diagram of the single PC12 cell release monitoring with the PPyox-CFE; (C) The obtained minimum amplitude of the spike.

Figure 6.

Stability of PPyox-CFE, expressed as percentage of sensitivity to DA in PBS (pH 7.4) and the detection limit of DA.