Rapid, inexpensive fabrication of electrophoretic microdevices for fluorescence detection

Abstract

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400-base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18-fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 ± 2 and 166.48 ± 3 bases, respectively.

Keywords: DNA; deoxyribonucleic acid; electrophoresis.

FIGURE 1

Inherent fluorescent emissions of candidate substrates for microfluidic device fabrication when excited by a 488‐nm sapphire laser: (A) signal from a sheet of polyethylene terephthalate (PeT), (B) toner‐coated PeT (tPeT), and (C) cyclic olefin copolymer (COC). Insets in (B) and (C) illustrate any change in the signal at 10 s when the substrate was placed on the custom electrophoresis system with a fluorescence detector

FIGURE 2

Prototype electrophoresis microchip architectures: (A) microchip with an “anchor”‐like cross‐T and a 4‐cm effective separation length (L _eff); (B) chip containing a 6‐cm L _eff and a traditional cross‐T design. Inset shows the cross‐T filled with polymer mixed with a blue dye for imaging purposes

FIGURE 3

Evaluation of the injection plug shape using fluorescein: (A) a series of images showing the injection of fluorescein in the “anchor”‐like cross‐T of the 4‐cm effective separation length (L _eff) chip; (B) the 6‐cm L _eff chip with fluorescein injected across the traditional cross‐T. For both conditions, −/+100 V were applied to the S and sample waste (SW) reservoirs for a 90‐s injection, then −200 V was applied to the B reservoir and +800 V was applied to the buffer waste (BW) reservoir, whereas both the S and SW reservoirs were ground

FIGURE 4

Electropherogram depicting separation of a 400‐base DNA size ladder showing differences in size as small as 5 bases and as large as 25 bases. Injection voltages of −100 and +100 V were applied to the S and sample waste (SW) reservoirs, respectively, for 90 s. Following injection, −200 V was applied to the B reservoir and +800 V was applied to the buffer waste (BW) reservoir, whereas both the S and the SW electrodes were ground. The inset table displays resolution (R _s) and base pair (bp) resolution (R _bp) calculated using PeakFit analysis software

FIGURE 5

Electropherogram from on‐chip electrophoresis completed in 10 min. Injection voltages of +/−100 V were applied to the S and sample waste (SW) reservoirs for 90 s, then −200 V was applied to the B and +800 V was applied to the buffer waste (BW) reservoir, whereas the S and SW reservoirs ground. PCR‐amplified DNA fragments (red) overlaid with a 400‐base DNA size ladder (black) (n = 3). The amplified fragments had expected sizes of 147 and 167 bases, respectively, and the average calculated size of each fragment was 148.62 ± 2 and 166.48 ± 3 bases (inset)