An immunoassay performed using a microchip electrophoretic system is described. Separation and quantitation of free and bound labeled antigen in a competitive assay are carried out in channels micromachined into fused silica substrates. Such microchips are attractive because of their small size, ruggedness, and amenability to automated handling. The assay achieves the determination of cortisol in blood serum over the range of clinical interest (1-60 micrograms/dL) without the need for extraction or other sample preparation steps. The separation is performed in less than 30 s. Very high throughput is possible by operating the assay in multiple channels in parallel. These characteristics make microchip electrophoretic systems a promising technology for the rapid analysis of clinical samples.

We present a model that quantitatively describes the performance of microfabricated electrophoretic devices filled with linear polyacrylamide as replaceable sieving material for single-stranded DNA analyses. The dependence of resolution on various separation parameters such as selectivity, diffusion, injector size, device length, and channel folding was investigated. A previously predicted dependence of longitudinal diffusion coefficient on electric field strength has been verified. We have used this model to develop and optimize microfabricated electrophoretic devices for DNA analyses. For single-color DNA sequencing mixtures, we routinely achieve separations of 400 bases in under 14 min at 200 V/cm, and separation of 350 bases in only 7 min at 400 V/cm, with a minimum resolution of R = 0.5. Our results also indicate reduced fragment biasing and efficient sample stacking for DNA sample loading on microfabricated devices.

We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microf luidic electrophoresis device. We have achieved baselineresolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci CSF1PO, TPOX, THO1, and vWA (abbreviated as CTTv) were performed in less than 2 min. This constitutes a 10-to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 m ؋ 100 m in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50°C. A f luorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 l was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.