To extend the duration of amplification, one could either tune Th and Tl by adjusting the thermal system or by designing specific primers with sufficiently high melting temperatures. The temperature at the interface between the oil and sample was measured using different sample volumes (Supplementary Figure S2). The results showed that the greater the volume of sample, the lower the Tl. Theoretically, one could add more sample volume to decrease the Tl and give a longer effective time. However, this could also stimulate a second mode of motion in natural convection if the fluid path were too long: that is, the flow patterns could split into two or more vertical circulation pathways. For even longer columns, the flow could become turbulent. These conditions should be avoided to ensure stability of the flow pattern. Moreover, increasing sample volume adds cost to the reaction by increasing the amount of PCR reagents required. Therefore, a fixed volume of 75 µL was chosen to maintain a stable, single-circulation thermal environment. The flow pattern in the 2.3 mm–diameter capillary tube was visualized using particle image velocimetry (PIV) (Figure 1C-III) and confirmed by three-dimensional FLUENT simulations (Figure 1C-IV; Supplementary Figure S3). The design described above solved the first challenge of flow generation and the temperature fields required for successful amplification.
For the second challenge, the optimal Tm values of primers under fixed thermal conditions should be tested: Th = 95°C at the bottom and Tl = 67°C at the oil–buffer interface. Seven primer sets with different Tm in a range of 57–80°C were designed to amplify hepatitis B virus (HBV) amplicons by CCPCR (Supplementary Table S2). The electrophoresis gel data (Figure 2A) show that although CCPCR did not amplify target DNA using primers with a Tm ≤ 70°C, primers with a Tm ≥ 72°C resulted in successful template amplification. Using primers with a Tm ≥ 76°C, the intensities of electrophoretic DNA bands appeared stronger.
Along with Tm–Tl, the temperature difference Th–Td for denaturation is the other important factor in CCPCR. If the Td is too close to the Th in the tubes, CCPCR will fail because the double-strand DNA is unlikely to have denatured completely, which will not allow primers to efficiently anneal to template DNA. This would diminish the ability of the platform to detect long amplicons since greater lengths or higher GC content of amplicons are usually accompanied by higher temperatures of denaturation. We tested four HBV amplicons (Supplementary Table S3) with a Td range of 86–91°C and found that CCPCR could amplify the two amplicons (122 and 169 bp) with Td <87°C (Th–Td >8°C), but failed to amplify the other two amplicons (188 and 222 bp) with Td >90 (Th–Td <5°C; Figure 2BI). To increase Th–Td, one can tune the heating temperature from 95°C to 99°C. Weak bands now appear for both the 188- and 222- bp amplicons with Td >90°C in Figure 2BII, indicating that a greater Th value can result in the successful amplification of DNA templates with higher Td values. It should be noted that the bands for both the 122- and 169-bp amplicons in Figure 2BII are weaker than those in Figure 2BI. The reason could be that the primer annealing efficiency for both of these amplicons with a Td ≤ 86°C is reduced by an increase in the oil-buffer interface temperature. One can also use chemical methods to reduce the Td of amplicons by adding dimethyl sulfoxide (DMSO), which enhances the denaturation of DNA secondary structures (23). However, a caveat is that DMSO also affects primer annealing on templates because the Tm of primers is lowered, which can adversely affect CCPCR amplification in certain cases (Figure 2BIII).
It is important to maintain the same thermal field (Th = 95°C; Tl = 67°C) for consistent flow and temperature distribution in the capillary tube, and it would be even better if one could choose amplicons with the desired Td without altering the Tm of primers. Selecting a low Td is a strategy for amplifying a long amplicon where there is the limitation that the heater temperature needs to be maintained at 95°C. For this reason, we attempted to amplify seven amplicons of different lengths, from 208 to 816 bp, using CCPCR. The Td of each amplicon was verified experimentally using melting curve analysis (data not shown). CCPCR could amplify lengths of DNA ≤500 bp (Figure 2C). This revealed the importance of Td as well as Tm in CCPCR, whereas traditional PCR relies more on Tm alone. In summary, a rule-of-thumb for CCPCR for any DNA template is to use a high-Tm primer and a low-Td amplicon. To demonstrate this rule, several different virus amplicons of ≤500 bp (Td ≤ 87°C, Tm ≥ 76°C) were chosen for testing and all were successfully amplified by CCPCR (Figure 3A). The sensitivity of the test with HBV plasmid DNA was 30 copies per reaction (Figure 3B). The high- and low-copy HBV DNA sequences (3000 and 30 copies/tube, respectively) were amplified by CCPCR in only 30 min (Figure 3C).