Journal
ELECTROPHORESIS
Volume 29, Issue 7, Pages 1431-1440Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/elps.200700470
Keywords
microfluidics; mitochondria; oversampling; quantile-quantile; undersampling
Funding
- NIA NIH HHS [R01 AG020866, K02-AG21453, R01 AG020866-06, AG20866, K02 AG021453-05, K02 AG021453] Funding Source: Medline
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This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 +/- 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile-quantile (q-q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial. events became too large (> 72 for rat liver and > 98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q-q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (< 55 for rat liver and < 81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.
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