Titer-plate formatted continuous flow thermal reactors: Design and performance of a nanoliter reactor

Abstract

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. Six different DNA fragments with lengths between 99 bp and 997 bp were successfully amplified at 1 mm/s. Repeatable, successful amplification of a 99 bp fragment was achieved with a minimum of 8000 copies of the DNA template. This is the first demonstration and characterization of continuous flow thermal reactors within the 8 mm × 8 mm footprint of a 96-well micro-titer plate and is the smallest continuous flow PCR to date.

Figure 1

(a) The physical dimensions of a 96 CFPCR array and a standard micro-titer plate; (b) A single CFPCR, 8 mm × 8 mm, including three distinct temperature zones compared to a penny.

Figure 2

(a) Boundary conditions for the model used to determine the physical dimensions of the fin needed to obtain sufficient cooling between the denaturation and renaturation zones, including the thermal and fluidic boundary conditions; (b) The temperature distribution in the PCR cocktail at the center of a microchannel for different fin heights, with a fin width of 400 μm.

Figure 3

The transition distance and the temperature gradient for different groove depths between the denaturation and renaturation zones.

Figure 4

(a) Boundary conditions for the thermal simulation for a single CFPCR device; (b) The simulated temperature distribution at the midplane of 20 microchannels with the ±2°C thermal contour lines, which also could be used to elucidate the temperatures of the 20 microchannels as a function of location on the chip.

Figure 5

Temperature distribution along the paths defined in Figure 4(b), which are located in the innermost microchannel.

Figure 6

(a) The boundary conditions used for the thermofluid model to estimate the temperature distribution along the center of the microchannel at different flow velocities; (b) The temperature distribution along a single microchannel at flow velocities from 0 mm/s to 4 mm/s.

Figure 7

The total time of each thermal cycle and the residence and transition times (s) for the PCR cocktail in each temperature zone as a function of flow velocity (mm/s). Isothermal heating zones are represented as solid segments and transitions between isothermal zones are shown filled with lines sloping up or down, depending on the direction of the temperature transition.

Figure 8

(a) Agarose gel image of amplicons from a commercial thermal cycler (Control), and the nanoliter CFPCR at linear flow velocities of 1 mm/s, 2 mm/s, and 3 mm/s. (b) The relative intensity of the amplification efficiency at each flow rate compared to the amplicon from a commercial thermal cycler (b) Amplification results for different DNA fragments from 99 bp, 125 bp, 150 bp, 200 bp, 500 bp, and 997 bp at a flow velocity of 1 mm/s (c) Amplification results for a 99 bp amplicon at different initial concentrations of template from 4.46×10⁻⁵ ng/μL to 4.46 ng/μL.