Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

Abstract

We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 +/- 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 +/- 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.

Figure 1

Sheath-flow cuvette. An array of 32 capillaries is held in a microfabricated sheath-flow cuvette. Sample migrates from the tips of the capillaries, forming thin filaments in the fluid-filled chamber. A single low-power laser beam simultaneously excites fluorescence from the sample streams.

Figure 2

Optical design of the instrument. A beam from a solid-state laser is focused into the sheath flow cuvette shown in Figure 1. Fluorescence is collected and collimated with an achromatic lens, filtered with an interference filter, and imaged onto a CCD camera with an identical achromatic lens.

Figure 3

Separation of sphingolipid standards using free solution electrophoresis in the 32 capillary instrument.

Figure 4

Capillary isoelectric focusing of pI 5.5 and 6.5 standards. The insert shows the raw data and the least squares regression analysis with a Gaussian peak (green) for the 6.5 pI standard. The standard deviation of the Gaussian function was 0.0021 pH units.

Figure 5

Capillary isoelectric focusing separation of myoglobin, ovalbumin, and pI 5.5 and 6.5 standards. The electropherograms were aligned based on the migration time of the standards, and the pH scale was based on the nominal pH of the standards.

Figure 6

Capillary isoelectric focusing of myoglobin components. The smooth curves are the data and the dashed curves are the least-squares regression fit of a pair of Gaussian functions to the peaks. The peaks were separated by 0.020 ± 0.001 pH unit.