In Vitro Electrochemistry of Biological Systems

Abstract

This article reviews recent work involving electrochemical methods for in vitro analysis of biomolecules, with an emphasis on detection and manipulation at and of single cells and cultures of cells. The techniques discussed include constant potential amperometry, chronoamperometry, cellular electroporation, scanning electrochemical microscopy, and microfluidic platforms integrated with electrochemical detection. The principles of these methods are briefly described, followed in most cases with a short description of an analytical or biological application and its significance. The use of electrochemical methods to examine specific mechanistic issues in exocytosis is highlighted, as a great deal of recent work has been devoted to this application.

Figure 1

Basic introduction to amperometric detection of exocytosis at single cells. (a) The top image is the typical setup for amperometry of a single cell. Exocytosis is stimulated by a pipette containing a stimulant, and the release is monitored by a carbon fiber electrode. The bottom image shows typical amperometric data. (b) The oxidation reaction for catecholamines. The catecholamine is oxidized to the orthoquinone form, losing two electrons. (c) Left-hand trace shows a series of stimulations, represented by arrows, and the electrochemical responses detected after each stimulation. Right-hand trace shows a single amperometric current transient. (d) Faraday’s equation, which is used to determine the amount of material released during exocytosis.

Figure 2

Measurements of serotonin overflow from enterochromaffin cells at adult or neonatal guinea pig ileum tissue. (a) Current versus time trace of serotonin overflow from adult guinea pig ileum tissue recorded with a diamond electrode (solid line) and carbon fiber electrode (dotted line). (b) Current versus time trace of serotonin overflow recorded with a diamond electrode for normal response (solid line) and in the presence of serotonin transporter (SERT) antagonist, fluoxetine (dotted line) for adult guinea pig ileum tissue. The current increased dramatically in the presence of fluoxetine, signifying that the measured current was indeed serotonin. (c) Current versus time trace of serotonin overflow recorded from neonatal guinea pig ileum tissue (dark blue) and the same tissue in the presence of fluoxetine (light blue). No difference in current profile was noted, suggesting that SERT activity in neonates is significantly low or underdeveloped as compared with adult. Abbreviations: OT_c, over tissue current; TT_c, touching tissue current. Panels a and b reproduced from Reference with permission. Panel c reproduced from Reference with permission.

Figure 3

Representative chronoamperometric recordings for serotonin uptake in brain stem synaptosomes prepared from serotonin transporter (SERT) +/+ (a), SERT +/− (b), and SERT −/− (c). The average uptake rates for SERT +/+ and SERT +/− were 162 ± 13 and 67.7 ± 4.1 pmol/mg protein-min, respectively, whereas SERT −/− showed no detectable uptake. Reproduced from Reference with permission.

Figure 4

Examples of confluent WSS cells electroporated with fluorescein diphosphate in stationary mode (a) and scanning mode (b, c). Five rings can be seen in image a, demonstrating the electrolyte-filled capillary’s ability to independently electroporate different places within one cell culture in the stationary mode. Fluorescent “e” and “snake” patterns in images b and c, respectively, were drawn using the scanning mode. Reproduced from Reference with permission.

Figure 5

Fast-scan cyclic voltammetry–scanning electrochemical microscopy images of a single RAW 264.7 macrophage cell following stimulation with zymosan particles (row 1), followed by the addition of 0.3 IU of catalase (row 2), and after washing with fresh Hank’s buffer (row 3). Column a shows the potential for reduction of O₂ average of 14 points between −1.2 and −1.3 V on cathodic sweep). Column b shows the potential for H₂O₂ oxidation (average of 12 points between +1.2 and +1.3 V on the first anodic sweep). Column c represents the average of −0.45 to −0.55 V on the cathodic sweep. The images in each row were recorded simultaneously. The potential applied to the tip consisted of a three-segment waveform (0.0 to +1.2 to −1.4 to 0.0 V) with a scan rate of 450 V·s⁻¹. Reproduced from Reference with permission.

Figure 6

(a) An optical image and (b) amperometric recordings of oxygen consumption obtained from a bovine embryo adhered to a microfluidic device integrated with four working electrodes (labeled W1, W2, W3, and W4 in the optical image). Panel a shows the immobilized embryo at the gate position; the plot of the oxygen concentration profile consumed by the day-6 embryo at the morula stage was obtained with the amperometric detector array at room temperature against time. The monitored concentration profiles for each electrode are shown in panel b. Reproduced from Reference with permission.