Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

Abstract

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 6 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.

Identifying indoor environmental patterns from bioaerosol material using HPLC

“…It is noted that higher resolving power strategies (other HPLC, GC and MS) were attempted, but detection was hampered by low species concentration—hence the strategy of overlapping peaks to boost signal strength coupled with pattern recognition. [25,26] The averaged HPLC trace displays three separate collections from the high-traffic indoor space and seven collections of the low-traffic indoor space. The standard deviation within each sample type between replicates was on average less than one absorbance unit in the raw chromatogram although this does scale with the normalization, and the average standard deviation over the information rich regions are much smaller than the separation between the two data sets.…”

Evaluation of peak overlap in migration-time distributions determined by organelle capillary electrophoresis: Type-II error analogy based on statistical-overlap theory

“…While this has been commonly assumed, our work justifies the validity of the assumption. However, we should not assume that peak overlap always will be negligible in organelle CE, particularly in CEs developed with chip technology producing relatively wide peaks [40] or CEs having high peak densities due to insufficient sample dilution or injection-volume overload [41]. Our simple procedure can be used to rapidly screen such CEs for peak overlap.…”

Analysis on micro-mixing enhancement through a constriction under time periodic electroosmotic flow

“…The measured values of fluorescein mobility in the literature can vary significantly from -0.3 9 10 -8 m 2 V -1 s -1 to -3.7 9 10 -8 m 2 V -1 s -1 Whiting et al 2008;Whang and Yeung 1992;Cheng et al 1990) at room temperature depending on pH, concentration and type of buffer solution. However, since the experimental conditions of the measured mobility value in the literature are different from our experiment, these values were not adopted in the simulation.…”

“…Incidentally, the configuration of our micromixer is similar to the conventional apparatus for measuring ion mobility with capillary electrophoresis (CE) (Wang et al 2007;Bahga et al 2010;Pumera et al 2002;Whiting et al 2008). Conventional CE system generally involves the injection of fluid plugs into a separation channel where the ions are separated based on their mobilities and the migration times of the ions can be determined.…”