We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

While performing routine electroosmotically driven CE separations on microfluidic chips, we have observed peak shape, migration time, and baseline drift anomalies. Pressure-driven backflow (opposing electroosmotic flow (EOF)) has been observed and characterized, and meniscus surface tension (Laplace pressure) is cited as the likely cause. However, there are a number of interdependent factors that affect bulk flow in a microchip environment, including evaporation, buffer depletion due to hydrolysis, EOF pumping, siphoning, viscosity changes due to Joule heating, and Laplace pressure. Given the complexity of such a system, pressure effects were isolated from EOF, and to some extent, siphoning effects were isolated from suspected meniscus effects. Pressure flow observed in the absence of an applied field ranged from 0.4 to 0.8 mm/s, which was on the order of the EOF generated experimentally, 0.6 mm/s at a field of 150 V/cm, and was some 10-20 times larger than what would be predicted merely from a difference in liquid levels (siphoning). Furthermore, experiments were performed without an electric field and with the chip tilted so that meniscus flow ran "uphill" against a siphoning backflow and showed siphoning flow to have a negligible effect upon meniscus flow under the microchip conditions studied. These findings are relevant to the profusion of microfluidic and array-based technology that also use microliter liquid volumes in like-sized reservoirs with similar menisci.

We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.

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A new convolution-detection method was developed which converts multiple-point (Shah function) detection, time-domain electropherograms into frequency-domain plots by means of a Fourier transformation, allowing the analytes' speeds to be viewed in terms of their "blinking" frequency; we have named this method Shah convolution Fourier transform detection, or SCOFT. This paper represents proof of principle of the detection concept. A micromachined glass stucture with a patterned layer of Cr on its top surface to form regularly spaced detection slits was used to perform capillary electrophoresis separations with 55-point, laser-induced fluorescence detection over 3.78 cm of the 6.6 cm separation channel. While this method can be easily integrated into a miniaturized total analysis system (μ-TAS), the principle is equally applicable to detection in full-sized analytical instrumentation. Single-component samples (fluorescein) migrating through the separation channel yielded a single peak in the frequency domain, and two-component samples (fluorescein and fluorescein isothiocyanate) yielded two resolved peaks, each at the expected frequency; harmonics were also observed. Advantages were seen in terms of isolation of the analyte peaks from interference such as baseline drift and line noise. Resolution is somewhat inferior to that seen in single-point detection, but it is thought that improved chip design and mathematical and instrument optimization will lead to performance superior to that of single-point detection.

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This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 × 9 array of gel posts each less than 1 μL in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures. PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added atop precast gel posts. PCR amplification occurred with template from purified DNA or from raw urine of patients with BK viruria. Multiple primer sets can be utilized per gel post array with no detectable cross contamination. As few as 34 BK virus templates were consistently detected by PCR in an individual gel post. Amplification of HPA1 and FGFR2 genes in human genomic DNA (gDNA) required as little as 2-5 ng of gDNA template/gel post. The device prototype includes a Peltier element for PCR thermal cycling and a CCD camera to capture fluorescence for product detection. Our technology is amenable to integration in point of care microdevices.

Sample preparation has long been recognized as a significant barrier to the implementation of macroscopic protocols on microfabricated devices. Macroscopically, such tasks as removing salts, primers and other contaminants are performed by methods involving precipitation, specialized membranes and centrifuges, none of which are readily performed in microfluidic structures. Although some microfluidic systems have been developed for performing sample purification, their complexity may hinder the degree to which they can be implemented. We present a method of microchip-based sample purification that can be performed with even the simplest microfluidic designs. The technique is demonstrated by removing primers from a sample of amplified DNA, leaving only the product DNA. This provides a new sample preparation capability for microfluidic systems.