Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device

Abstract

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.

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

“…Integrating equation (1), we obtain the total diffusion flux of vapor at any given moment, (2) where x is the diffusion length (i.e., the distance between the sample interface and the loading holes); p 0 and p v are the ambient and sample interfacial vapor pressures, respectively. Assuming a pseudo-steady-state condition (i.e., the sample interface recedes slowly), the diffusion flux also equals the amount of water leaving the liquid meniscus, (3) where M w and ρ w are the molecular weight and density of water, respectively. Combining equation (2) and (3), and integrating from t = 0 to t = t F , we obtain the distance from the sample interface to the sample access hole as a function of time at a constant temperature: (4) where x 0 , x F are the initial and final diffusion lengths, respectively; T i is sample interfacial temperature; and t F is the reaction time.…”

“…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.…”

A miniature integrated device for automated multistep genetic assays

“…These complexities have limited reports of integrated reactions to a single reaction combined with analysis (3,4). To achieve full integration we have employed batch reactions, sensorless reagent positioning and careful choice of materials.…”

“…Recent applications of robotics can improve consistency and reduce labor but call for dedicated personnel and are susceptible to contamination. Previous efforts towards integration and miniaturization have addressed only one or two operations at a time (1)(2)(3)(4).…”

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.…”