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 +/- 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.

Figure 1

Schematic diagram of microfluidic devices and orthogonal fluorescence detection scheme. (A) 1, 2, 3, and 4 represent the sample, sample waste, buffer, and buffer waste reservoirs, respectively, with a detection zone 36.0 mm from the channel intersection. All distances are given in mm. (B) Channel dimensions as defined by the etch depth (r_e = 20 µm) and the mask width (m = 10 µm). (C) Laser light is focused onto the microfluidic device with a 6.3× objective (FO), fluorescence is collected with a 60× long working distance objective (CO), passed through a 505 nm long pass filter (LP), a 535 ± 17 nm bandbass filter (BP), and a pinhole (P) before being detected by the photo-multiplier tube (PMT). The pinhole produces a collection zone (r_p) defined by the position of the pinhole in the detector. Alternately, a mirror (M) could redirect the fluorescence to an eyepiece (EP) for rough alignment. The pinhole is placed at either 47 or 66 mm from the back of the collection objective so that light is collected from a zone on the device with a radius of either 27.8 or 19.8 µm.

Figure 2

Electropherogram of mitochondrial events separated on a glass microfluidic device with PVA dynamic coating buffer. (A) 55 rat liver mitochondrial events and (B) 82 143B mitochondrial events. Insets are a histogram of the electrophoretic mobilities of the events in each figure

Figure 3

q–q plots of mitochondrial distributions versus the average of the quantiles from the unbiased distributions. (A) Unbiased rat liver, (B) unbiased 143B, (C) oversampled rat liver, (D) oversampled 143B, (E) undersampled rat liver, and (F) undersampled 143B.