Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules

Abstract

Microfluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the laboratory environment. In closed systems however, the high temperatures of up to 95 °C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23 °C to 95 °C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.

A microfluidic timer for timed valving and pumping in centrifugal microfluidics

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Centrifugal microfluidic platforms: advanced unit operations and applications

“…The application of this unit operation was demonstrated for PCR amplification and subsequent transport of the amplification product for further analysis. 176 Recently, Focus Diagnostics and 3M introduced the integrated cycler, a real time PCR cycler, to the market. Up to 96 pre-extracted nucleic acid samples can be pipetted to a universal single-use disk.…”

Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation

“…The pre-amplification chamber itself constitutes a structure for centrifugo-dynamic pumping 69 and is connected with a capillary to an auxiliary gas chamber. The capillary provides a vapor diffusion barrier and, in combination with the auxiliary gas chamber, is employed to significantly reduce pressures in closed chambers during thermocycling as described in detail in previous work 70 . Under constant rotation of 30 Hz, the consensus multiplex PCR pre-amplification is initiated by a polymerase activation step as the LabDisk-Player heats the liquid compartment to 95°C for 2 min, followed by 12 thermocycles (20 s at 95°C, 60 s at 52°C and 30 s at 70 C).…”