Laser-ablated poly(methyl methacrylate) microdevices for sub-microliter DNA amplification suitable for micro-total analysis systems

Abstract

Biochemical techniques, such as the polymerase chain reaction (PCR), can take up to 3.5 h for completion using a commercial bench-top instrument, creating a bottleneck in sample preparation processes. PCR has been successfully adapted to microfluidic devices, reducing the time needed to as little as 7-10 min. Recently, a trend in the field is to use alternative substrates, such as poly(methyl methacyrlate) (PMMA), for the fabrication of microfluidic devices. PMMA has several advantages over more expensive substrates including rigidity without fragility, disposability, and it is easy to fabricate, using techniques such as hot embossing or CO 2 laser ablation. Here, we report the fabrication of PMMA microdevices to explore their effectiveness for PCR amplification. Several types of PMMA microdevices were fabricated using a CO 2 laser ablation system, with two or three PMMA layers of different thicknesses. Bonding of the microdevices was significantly improved through the use of a grid system etched into the device, allowing for trapped air to escape, eliminating leakage. Using infrared thermal cycling, the ramping rates were determined to be dependent on the thickness of the PMMA used in fabrication, allowing for customization of cycling conditions. Further reduction of the thermal mass by isolation of the chambers provided a significant increase in the heating and cooling rates (up to 6.19 ( ± 0.32 • C s −1 ) and −7.99 ( ± 0.06 • C s −1 ), respectively). Bubble formation, a chronic problem in microfluidic systems in general and problematic during the heating phase of PCR, was minimized through the use of a biocompatible adhesive/manifold combination to seal the reservoirs. Finally, successful PCR amplification was demonstrated with both a fragment from the β-globin gene and 15 tetra-nucleotide repeat regions with a sex-typing marker in a conventional STR kit with the latter facilitated by the dynamic coating of the fluidic architecture with poly(ethylene glycol) (PEG).

Fast and versatile fabrication of PMMA microchip electrophoretic devices by laser engraving

“…Although traditionally limited by the resolution of the engraver, the process has been explored to fabricate microfluidic components for different applications including injectors [28], channels [29,30], pumps [31], mixers [32], cytometers [33], valves [34], reactors [35], and devices for PCR amplification [36].…”

“…Laser engraving of PMMA is typically accomplished using a CO 2 laser beam to break bonds in the polymer surface and remove the decomposed material from the ablated regions by the combination of photochemical and photothermal processes (melting, vaporization, and decomposition) [22]. Although traditionally limited by the resolution of the engraver, the process has been explored to fabricate microfluidic components for different applications, including injectors [28], channels [29,30], pumps [31], mixers [32], cytometers [33], valves [34], reactors [35], and devices for PCR amplification [36].…”

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis

“…4(b). Microbubble formation and evaporative loss during on-chip PCR are known issues 44 and developing an effective containment system for the PCR solution during thermal cycling is of critical importance. Starting with 10 mL of metered elution buffer, the volume recovered post-PCR was increased from 80 to 95%, with a 69% reduction in the standard deviation.…”

Fast and versatile fabrication of PMMA microchip electrophoretic devices by laser engraving

“…Although traditionally limited by the resolution of the engraver, the process has been explored to fabricate microfluidic components for different applications including injectors [28], channels [29, 30], pumps [31], mixers [32], cytometers [33], valves [34], reactors [35], and devices for PCR amplification [36]. …”