PCR, chamber

Lagally et al. [74] developed an integrated device for PCR and NCE with electric field control and fluorescence detection. Furthermore, the device had integrated heaters and temperature sensors, as well as PDMS membrane valving to control analyte transport. The PDMS unit was fabricated as membrane valves between a glass PCR chamber and the glass NCE channel, allowing precise control of both process unit operation and analyte transport. 

Figure 8.22. Capillary electrophoresis on a chip, (a) Schematic of the microchip used for PCR amplification and electrophoresis. The direction of arrows indicate injection (I) and separation (S). (b) Electrophoretic microchip with multiple PCR chambers.

Integration of sample preparation and analysis [46] is one of the prime objectives of /i-TAS. PCR on a chip is one of the earliest applications of sample preparation. It has been carried out in the sample reservoir of the electrophoretic chip shown in Figure 8.22a. The nucleotides, primers, and other chemicals are added into the sample reservoir, and the entire device is introduced into a conventional PCR thermal cycler. The PCR products from the sample reservoir are then injected into the separation channel and analyzed. A more complex chip with multiple PCR chambers is shown in Figure 8.22 b. 

The polymerase chain reaction is the prevalent method for DNA amplification. Much effort has been made to integrate PCR chambers on microchips to carry out amplifications of DNA molecules prior to their analysis. For instance, PCR was first achieved on a Si-based reaction chamber (25 or 50 pL) integrated with a polysilicon thin-film (2500-A-thick) heater for the amplification of the GAG gene sequence (142 bp) of HIV (cloned in bacteriophage M13) [997]. 

On-chip PCR benefits from the high surface-to-volume ratio (SVR) of the PCR chamber. For instance, SVR of Si chambers are 10 mm2/pL [916] and 17.5 mm2/pL [917], which are greater than 1.5 mm2/pL in conventional plastic reaction tubes and 8 mm2/pL in glass capillary reactive tubes [917,918]. 

In one report, a thin-walled PCR chamber was constructed on a PET chip. The thin membrane (200 pm) between the chamber and the A1 heater resulted in... 

In a later report, inhibition of PCR in Si-glass PCR chips was found to be mainly caused by the adsorption of the Taq polymerase, rather than by the adsorption of DNA [299]. It was also found by XPS analysis that the primary PCR inhibitor in a glass PCR chamber was Cr, which was involved in the microfabrication process. The highest Cr concentration that could be tolerated was found to be 0.1 M [932]. 

FIGURE 9.8 Temperature profile of the PCR chamber (center) fabricated on different substrates silicon (solid line), glass (broken line), PDMS with bottom heating (dotted line), and PDMS with both top and bottom heating (chain line). Top inset shows the temperature profile in the expanded scale (90-94°C) [1000]. Reprinted with permission from Institute of Physics Publishing. 

Multiple PCR chambers have been fabricated on a single microfluidic chip and explored for high throughput PCRs [78-83]. An example of a multichamber micro-PCR device, the micro-DNA amplification and analysis device, (p-DAAD) consisted of 16p-DAADs in parallel with each p-DAAD consisting of four microreactors fabricated on a 4" silicon wafer (see Fig. 4). Multichamber micro-PCR devices [84] have been demonstrated for DNA amplifications of five gene sequences related to E. coli from three different DNA templates and detected by TaqMan chemistry with a limit of detection (LOD) of 0.4 copies of target DNA. 

Fig. 16 (Continued) (e) PCR chamber with exit channel tapering before intersecting with the MR inlet channel (Scale bar. 1 mm), (f) Cross-T intersection (Scale bar 1 mm). The relative sizes of the BR, SW, and BW channels create the difference in volume displacement during the pumping injection and affect how the resistance is dropped under an applied separation voltage. Reproduced from [10] with permission...

Fig. 19 Mask design for the portable PCR-CE system. The glass mlcrocharmels are indicated in black, the RTD and microfabricated electrodes are in green, and the heater (located on the back of the device) Is shown in red. The PCR chamber is loaded through reservoirs a and b. Reservoir c is the co-inject reservoir, d is the cathode, e is the waste, and/is the anode. Reproduced from [ill] with permission...

Fig. 20 Representative nine-plex STR profiles of (a) 9,947A female and (b) 9,948 male standard DNA obtained with 100 copies of DNA template in the PCR chamber of an integrated microfluidic system. Reproduced from [258] with permission...

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