Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

Abstract

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.

Effect of buffer flow on DNA separation in a microfabricated electrophoresis system

“…4B. The error bars are based on detection at four different points (4,8,12, and 16 mm) along the channel. This phenomenon confirms the ion compensation effect by the increase of buffer concentration, although it is different from the common understanding of ionic effect on DNA migration in which the mobility is inversely proportional to the square root of the ionic strength in the gel [28].…”

“…Other components, such as temperature control, electrode arrays, and optical detection are also critical in making a fully functioning self-contained microbioanalysis system. Si/glass devices provide an ideal platform for integrating components of diversified functionalities in a relatively small area [8][9][10]. With either internal or external components, many devices are capable of performing separation and detection for portable genomic analysis.…”

An integrated microfluidic chip for DNA/RNA amplification, electrophoresis separation and on‐line optical detection

“…The detection of Escherichia coli O157 and Salmonella typhimurium was successfully demonstrated using the proposed design. Similarly, Rodriguez et al [21] developed an integrated PCR-CE system for genetic analysis. In their study, the PCR chamber was fabricated using silicon and it incorporated aluminum heaters and temperature sensors.…”

Microfabricated valveless devices for thermal bioreactions based on diffusion-limited evaporation

“…A miniaturized bioreaction system presents several advantages over the bench-top equivalent: reduced reagent, labor and equipment costs, decreased reaction time, reduced risk of contamination, and simplified sample handling. There are two major types of miniaturized bioreaction systems: batch-based systems where the stationary reaction solution is heated or thermocycled inside a reaction chamber by either external heaters [3][4][5][6][7][8][9][10][11][12][13][14][15][16] or integrated on-chip heaters, [17][18][19][20][21][22][23][24] and continuous flow-based systems where the sample flows through certain temperature zones with well-defined flow rates. [25][26][27][28][29][30] Other novel approaches, such as on-chip rotary reaction, 31 noncontact infrared-mediated reaction, [32][33][34] electrokinetically synchronized reaction, 35 electrowetting-based reaction 36 and Rayleigh-Bénard convection-based reaction 37,38 have also been reported.…”

“…Recent trends in miniaturized bioreaction systems are to integrate bioreactions with sample preparation, fluidic handling, and product detection to produce systems that can rapidly, conveniently, and economically extract information from raw biological samples with greatly reduced cost. 4,7,11,12,[17][18][19][20]23,39 One technical challenge in miniaturizing bioreaction systems is preventing or reducing evaporative loss during thermocycling. Although mineral oils, [4][5][6][7][8][9][10][11][12][13][14][15][16]10,26 adhesive tapes 19,22 and silicone rubber gaskets 21 have all been used in miniaturized bioreaction devices, most integrated systems reported so far have used microvalves to prevent evaporative loss.…”