Ultrafast Microfluidic PCR Thermocycler for Nucleic Acid Amplification

Abstract

The polymerase chain reaction (PCR) is essential in nucleic acid amplification tests and is widely used in many applications such as infectious disease detection, tumor screening, and food safety testing; however, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limit their application in special scenarios such as hospital emergencies, airports, and customs. Here, we propose a temperature control strategy to significantly increase the ramp rates for the solution temperature by switching microfluidic chips between multiple temperature zones and excessively increasing the temperature difference between temperature zones and the solution; accordingly, we have designed an ultrafast thermocycler. The results showed that the ramp rates of the solution temperature are a linear function of temperature differences within a range, and a larger temperature difference would result in faster ramp rates. The maximum heating and cooling ramp rates of the 25 μL solution reached 24.12 °C/s and 25.28 °C/s, respectively, and the average ramp rate was 13.33 °C/s, 6–8 times higher than that of conventional commercial PCR devices. The thermocycler achieved 9 min (1 min pre-denaturation + 45 PCR cycles) ultrafast nucleic acid amplification, shortening the time by 92% compared to the conventional 120 min nucleic acid amplification, and has the potential to be used for rapid nucleic acid detection.

Assessing Different PCR Master Mixes for Ultrarapid DNA Amplification: Important Analytical Parameters

“…Typically, the Taq-based DNA extension speed in PCR is ~60–100 base-pairs/s [ 14 ], usually measured through rapid deletion mutants of Taq (KlenTaq) [ 15 ], which are missing 5′-3′ exonuclease activity [ 2 , 16 , 17 ]. This hydrolyzing 5′-3′ Taq activity is a prerequisite for TaqMan probe-like assays, but the drawback of using these enzymes is the need for longer annealing/extension times.…”

“…The above results indicate that the PCR assay can be accomplished in 10 min for 40 cycles with a high PCR sensitivity and impressive reproducibility among replicate experiments, within the same PCR reaction chamber. With the nested PCR approach used by Bio Fire-like protocols [ 20 ] and further design miniaturizations [ 2 , 13 , 20 , 21 , 22 , 23 , 24 , 25 ], this PCR could have impressive multiplexing capacity without any significant changes in biochemistry.…”

“…These techniques have applications in fields such as clinical diagnostics, pathogen detection, and genotyping, where rapid turnaround times are crucial. Ultrarapid PCR techniques aim to provide results in a fraction of the time compared to traditional PCR methods [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ].…”

Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection

“…The real-time PCR device was designed and improved based on the previous work of the research group. 43 The most significant changes were the addition of two temperature zones for more flexible temperature control, the addition of a photoelectric detection module compatible with the amplification module for real-time fluorescence detection, and the in-depth analysis of the critical factors affecting the speed of liquid heating and cooling and detection sensitivity, demonstrating its enormous potential for rapid nucleic acid detection.…”

Rational PCR Reactor Design in Microfluidics

“…This system executed 30 RT-PCR cycles in 7.5 min with regular microliter-sized samples (25 µL) and a 0.03 °C thermalization homogeneity. Second is the reactor by Yi-Quan et al, using a static shuttling reactor, achieved a γ of 9 min/25 µL = 0.3, using a 50 µm polypropylene reactor wall and using pressure on the heater to heat spreader film and pressure and silicon grease between the heat spreader films and the reactor chamber [ 45 ]. Both efforts demonstrate that the best way to improve a static thermal-diffusion-only reactor is by minimizing the thermal contact resistance.…”