Integrated System for Rapid PCR-Based DNA Analysis in Microfluidic Devices

Abstract

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.

Surface modification in microchip electrophoresis

“…This is especially important for glass devices where significant adsorption is often observed at unmodified surfaces. An integrated microchip system for PCR reaction and DNA separation on glass substrates was reported by Khundurina et al [52]. Dynamic coating was applied prior to each analysis using a commercial separation buffer from Bio-Rad (CE-SDS Protein Kit) containing a proprietary agent in order to suppress the EOF.…”

“…A benefit of MCE compared to classical CE is the possibility to realize complex structures such as microfluidic networks on chip which enables to integrate chemical reactions as well. Interaction of reagents with the surface can, however, impair the intended chemical reaction which is especially true for one of the most attractive processes the polymerase chain reaction (PCR) [27].…”

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.…”

“…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. These microvalves seal the reaction chamber using pneumatically or mechanically actuated diaphragms, [7][8][9]11,15,[17][18][40][41][42][43] thermally actuated phase-change pistons, 12,19,39,44 or polyacrylamide gels.…”

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

“…The most common detection scheme requires capillary electrophoretic separation of the PCR product (along with molecular weight markers), followed by laser-excited fluorescence detection. [16][17][18][19] However, optical systems are difficult to miniaturize onto a monolithic microanalytical system. Towards the goal of achieving a fully integrated DNA microchip, alternative microfabrication-compatible detection techniques have to be sought for.…”