The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four color detection channels. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in < 120 s with the microplate system. The practical utility and multicolor detection capability is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a noncovalent 2-color staining method. This work establishes the feasibility of performing high-throughput genotyping separations with capillary array electrophoresis microplates.

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Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal f luorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and f luorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Turns in microfabricated capillary electrophoresis channels generally result in degraded separation quality. To circumvent this limitation, channels were constructed with different types of turns to determine the design that minimizes turn-induced band broadening. In particular, tapered turns were created by narrowing the separation channel width before the start of a turn and widening the channel after the turn is complete. The radius of curvature of the turn, the length over which the channel is tapered, and the degree of tapering were explored. The column efficiencies were determined by examining the resolution of the 271/281 base pair doublet in the separation of a phiX174 HaeIII DNA sizing ladder. Tapered turns with the smallest radius of curvature (250 microm), the shortest tapering length between the separation and turn widths (55 microm), and the largest tapering ratio (4:1 separation channel width to turn channel width) produced the highest resolution separations. These results are discussed by comparison to theoretical predictions of the effect of tapers and turns on analyte band dispersion in capillary electrophoresis.

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