Spatially and temporally resolved single-cell exocytosis utilizing individually addressable carbon microelectrode arrays

Abstract

We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-mum-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 microm) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors.

Figure 1

Schematic drawing of carbon fiber MEAs containing two (A), three (B), and seven (C) microdisks.

Figure 2

Scanning electron microscopy of carbon fiber MEAs having two (A), three (B), and seven (C) microdisks.

Figure 3

Steady-state voltammetric response of a two-fiber MEA in 1 mM FcCH₂OH containing 0.2 M KCl at a scan rate of 10 mV/s; (a) the response of microelectrode a alone, (b) the response of microelectrode b alone, and (c) the response of both microelectrodes operated and connected together.

Figure 4

Simulated distribution of steady-state diffusive flux at the cross section of a 5-μm two-fiber MEA as a function of the interelectrode distance. The d/a are (A) 2.4, (B) 4, (C) 10, and (D) 20, respectively.

Figure 5

Steady-state voltammetric response at 20 mV/s of a seven-fiber MEA in 1 mM FcCH₂OH and 0.2 M KCl. (A)—(G) are the voltammetric response of individual microelectrodes A—G as shown in the schematic of the microelectrode assemble shows in the bottom right.

Figure 6

Fast-scan voltammetric response (300 V/s) of the same seven-fiber MEA in 1 mM FcCH₂OH and 0.2 M KCl. (A)—(G) are the voltammetric response of individual microelectrodes A—G (as seen in Figure 5).

Figure 7

(A) Optical image showing a microelectrode array positioned over a single PC12 cell (right) and a stimulation pipet (left). The cell is denoted with an arrow. (B) Amperometric traces of exocytotic release from a PC12 cell recorded using a microelectrode array. Thick black lines along the time axis indicate exposure to high potassium stimuli (100 mM, 5-s pulse every 45 s). (C) The average value of normalized number of exocytotic events recorded with each electrode in the array. Eight different microelectrode arrays were use to examine 16 PC12 cells. Error bars are (S.E.M.

Figure 8

A 1-s time period of an exocytotic response of a PC12 cell after potassium stimulation showing simultaneous detection of concurrent events at different locations on the same cell. Red arrows indicate these events.