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. 2009 Feb 21;9(4):606-12.
doi: 10.1039/b807915c. Epub 2008 Nov 18.

A disposable, self-contained PCR chip

Jitae Kim  1 , Doyoung Byun, Michael G Mauk, Haim H Bau
Affiliations

A disposable, self-contained PCR chip

Jitae Kim et al. Lab Chip. .

Abstract

A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just-on-time releasable, paraffin-passivated, dry reagents is described. During both storage and sample preparation, the paraffin immobilizes and protects the stored reagents. Fluid flow through the reactor leaves the reagents undisturbed. Prior to the amplification step, the chamber is filled with target analyte suspended in water. Upon heating the PCR chamber to the DNA's denaturation temperature, the paraffin melts and moves out of the way, and the reagents are released and hydrated. To better understand the reagent release process, a scaled up model of the reactor was constructed and the paraffin migration was visualized. Experiments were carried out with a 30 microl reactor demonstrating detectable amplification (with agarose gel electrophoresis) of 10 fg ( approximately 200 copies) of lambda DNA template. The in-reactor storage and on-time release of the PCR reagents reduce the number of needed operations and significantly simplifies the flow control that would, otherwise, be needed in lab-on-chip devices.

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Figures

Figure 1
Figure 1
A schematic of the PCR chip with pre-stored, wax-encapsulated, dry reagents. (A) cross-section of a reactor. (B) An exploded view of the chip comprised of three layers of polycarbonate sheets (the thicknesses of layers I, II, and III are, respectively, 250μm, 750μm, and 250μm). (C) A photograph of the PCR chip containing two PCR reactors.
Figure 2
Figure 2
Visualization of the migration of the wax initially coated along (A) the ceiling and (B) the floor of a polycarbonate chamber. The dyed water appear dark and the wax is whitish. The chamber was held in a vertical position and heated on a hot plate maintained at 95 °C. The dotted lines identify the contour of the wax patch.
Figure 3
Figure 3
Schematics of (A) the initial and (B) the final position of the paraffin
Figure 4
Figure 4
A schematic diagram of the thermoelectric-based, thermocycler setup. The PCR micro reactor is sandwiched between two Peltier units controlled with a LabVIEW™ program.
Figure 5
Figure 5
The bottom (solid line) and top (dashed line) temperatures as functions of time during the cycling process when the reactor is controlled with two Peltier units
Figure 6
Figure 6
Instantaneous, dimensionless temperature distribution along the reactor’s height at various times t = 0, 0.5, 1, 1.5, 2, 2.5 and 4 s in the absence (solid line) and presence (dashed line) of a paraffin layer. The subscripts B, W, P, and T denote, respectively, the bottom polycarbonate layer, the water layer, the paraffin layer, and the top polycarbonate layer.
Figure 7
Figure 7
Agarose gel (1.5 %) electrophoresis images of PCR products amplified from a 10fg lambda DNA template. All the PCR amplifications were carried out in the polycarbonate reactor (35 cycles). Lane M is a marker VIII ladder. Lanes 1 and 2 correspond, respectively, to the products of the PCR reactor with the paraffin-encapsulated reagents and the PCR reactor operating with wet reagents.
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