Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection.
The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.
Duffy
Ciarán F
CF
National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland. ciaran.duffy@abbott.com
MacCraith
Brian
B
Diamond
Dermot
D
O'Kennedy
Richard
R
Arriaga
Edgar A
EA
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary; D046209:Electrophoresis, Microchip; D005453:Fluorescence; D007834:Lasers; D008930:Mitochondria, Liver