Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC-68 uC) zones; above 6 mm s 21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s 21 to 20 mm s 21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle 21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle 21 ). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).

Continuous flow polymerase chain reactors (CFPCRs) are BioMEMS devices that offer unique capabilities for the ultra-fast amplification of target DNA fragments using repeated thermal cycling, typically over the following temperature ranges: 90 degrees C-95 degrees C for denaturation, 50 degrees C-70 degrees C for renaturation, and 70 degrees C-75 degrees C for extension. In CFPCR, DNA cocktail is pumped through the constant temperature zones and reaches thermal equilibrium with the channel walls quickly due to its low thermal capacitance. In previous work, a polycarbonate CFPCR was designed with microchannels 150 microm deep, 50 microm wide, and 1.78 m long-including preheating and post-heating zones, fabricated with LIGA, and demonstrated. The high thermal resistance of the polycarbonate led to a high temperature gradient in the micro-device at steady-state and was partly responsible for the low amplification yield. Several steps were taken to ensure that there were three discrete, uniform temperature zones on the polycarbonate CFPCR device including: reducing the thickness of the CFPCR substrate to decrease thermal capacitance, using copper plates as heating elements to ensure a uniform temperature input, and making grooves between temperature zones to increase the resistance to lateral heat conduction between zones. Finite element analyses (FEA) were used to evaluate the macro temperature distribution in the CFPCR device and the micro temperature distribution along a single microchannel. At steady-state, the simulated CFPCR device had three discrete temperature zones, each with a uniform temperature distribution with a variation of +/-0.3 degrees C. An infrared (IR) camera was used to measure the steady-state temperature distribution in the prototype CFPCR and validated the simulation results. The temperature distributions along a microchannel at flow velocities from 0 mm/s to 6 mm/s were used to estimate the resulting temperatures of the DNA reagents in a single microchannel. A 500 bp DNA fragment was generated from a bacteriophage lambda-DNA target using 20 cycles of PCR. The amplification efficiencies compared to a commercial thermal cycler were 72.7% (2 mm/s), 44% (3 mm/s), and 29.4% (4 mm/s). The amplification efficiency with the modified CFPCR device increased by 363% at 2 mm/s and 440% at 3 mm/s compared to amplification obtained using a CFPCR device with the same fluidic layout, (Hashimoto et al., Lab Chip 4:638, 2004) strictly due to the improved temperature distribution.

No abstract

Surfactant and budesonide enhance the pulmonary distribution of fluorescent dye in mice.

Arrays of continuous flow thermal reactors were designed, configured, and fabricated in a 96-device (12 × 8) titer-plate format with overall dimensions of 120 mm × 96 mm, with each reactor confined to a 8 mm × 8 mm footprint. To demonstrate the potential, individual 20-cycle (740 nL) and 25-cycle (990 nL) reactors were used to perform the continuous flow polymerase chain reaction (CFPCR) for amplification of DNA fragments of different lengths. Since thermal isolation of the required temperature zones was essential for optimal biochemical reactions, three finite element models, executed with ANSYS (v. 11.0, Canonsburg, PA), were used to characterize the thermal performance and guide system design: (1) a single device to determine the dimensions of the thermal management structures; (2) a single CFPCR device within an 8 mm × 8 mm area to evaluate the integrity of the thermostatic zones; and (3) a single, straight microchannel representing a single loop of the spiral CFPCR device, accounting for all of the heat transfer modes, to determine whether the PCR cocktail was exposed to the proper temperature cycling. In prior work on larger footprint devices, simple grooves between temperature zones provided sufficient thermal resistance between zones. For the small footprint reactor array, 0.4 mm wide and 1.2 mm high fins were necessary within the groove to cool the PCR cocktail efficiently, with a temperature gradient of 15.8°C/mm, as it flowed from the denaturation zone to the renaturation zone. With temperature tolerance bands of ±2°C defined about the nominal temperatures, more than 72.5% of the microchannel length was located within the desired temperature bands. The residence time of the PCR cocktail in each temperature zone decreased and the transition times between zones increased at higher PCR cocktail flow velocities, leading to less time for the amplification reactions. Experiments demonstrated the performance of the CFPCR devices as a function of flow velocity, fragment length, and copy number. A 99 bp DNA fragment was successfully amplified at flow velocities from 1 mm/s to 3 mm/s, requiring from 8.16 minutes for 20 cycles (24.48 s/cycle) to 2.72 minutes for 20 cycles (8.16 s/cycle), respectively. Yield compared to the same amplification sequence performed using a bench top thermal cycler decreased nonlinearly from 73% (at 1 mm/s) to 13% (at 3 mm/s) with shorter residence time at the optimal temperatures for the reactions due to increased flow rate primarily responsible. SixPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access