Flow thermal cycler

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Flow-through thermal cycler (also called flow thermal cycler ) is a thermal cycler for continuous operation. It is used in the polymerase chain reaction (PCR).

principle

Since the handling of the smallest sample quantities when filling and emptying stationary chip thermocyclers is impractical, the principle of the flow thermocycler was developed. In contrast to the stationary thermal cyclers, in these reactors the reaction liquid is repeatedly moved through several areas of different temperatures, while the local temperatures in the various areas are kept constant. The temperature cycles of the PCR come about because the process liquid periodically passes through different temperature zones. The number of cycles is determined by the number of passages through the temperature zones. Flow PCR systems usually have three temperature zones.

Designs

A number of different embodiments have been developed for flow-through PCR. Most flow thermal cyclers can, however, be assigned to a few type classes that differ in the type of liquid flow: In the first variant, the channel lies as a meander within one level. Such an arrangement has the disadvantage that, in temperature programs with three or more temperature levels, the middle temperature level must be passed through both during heating and cooling. However, it has the advantage that it is well compatible with planar technology , so that such thermal cyclers can be easily manufactured in chip format.

An alternative to this is a helical (helical) guidance of the reaction channel. In this variant, a sense of direction can be implemented in the periodic sequence of temperatures. Such an arrangement can be easily implemented using a capillary tube. In contrast, this variant is unsuitable for planar production because of the crossing of the channels.

The arrangement of the liquid channel in a flat spiral represents a compromise between the two variants. With such a component, only the centrally located fluid connection has to be led out of the spiral plane. With this arrangement, the temperature zones can be formed by sectors of a circle . The disadvantage of this variant is that, given a constant channel cross-section in the spiral, different dwell times result in the individual cycles, or a variable channel cross-section has to be used.

Serial flow thermal cyclers

If flow-through thermal cyclers are operated with homogeneous fluids, the diffusion leads to a broad distribution of the reactants' residence time, which has an unfavorable effect on the efficiency of the process and is reflected in the PCR in chain breaks. However, very narrow residence time distributions can be achieved by using microfluid segment technology. The column of liquid is broken down into small segments (drops), which are transported like a plug. Such a segmentation of an aqueous fluid can e.g. B. caused by a mineral oil or liquid perfluorinated hydrocarbons as a carrier medium, since these are not miscible with the aqueous phase. Drop-based micro-flow processes, such as the drop-based flow PCR, also offer the advantage that many samples can be sent through the reactor one after the other without the samples being mixed. This enables high sample throughputs to be achieved in serial operation with constant process conditions.

application

Miniaturized flow-through thermal cyclers are used as components of miniaturized DNA analysis systems. In these systems, the micro-total analysis principle (µ-TAS) is transferred to DNA analysis. Such analysis systems are of interest for mobile medical diagnostics, for the identification of microbial pathogens , for forensic examinations and for the origin and quality control of food.

Individual evidence

  1. ↑ V. Baier et al., DE 4435107 C1 (September 30, 1994 / April 4, 1996)
  2. ↑ MU Kopp et al., Science 280 (1998), 1046-1048
  3. ↑ I. Schneegass et al., Lab Chip 1 (2001), 42-49
  4. ↑ CS Zhang et al., Biotechnol. Adv. 24 (2006): 243-284
  5. ↑ M. Curcio et al., Anal. Chem. 75: 1-7 (2003)
  6. ↑ R. Hartung et al., Biomed. Microdev. 11: 685-692 (2009)
  7. ↑ W. Wu et al., Analyst 140 (2015), 1416-1420
  8. ↑ CJ Easley, PNAS 103 (2006), 19272-19277
  9. ↑ T. Vilkner et al., Anal. Chem. 76: 3373-3385 ​​(2004)
  10. ↑ BH Park et al., Biosensors and Bioelectronics 91 (2017), 334-340
  11. ↑ CG Koh et al., Anal. Chem. 75: 4591-4598 (2003)
  12. ↑ Peham, JR, et al., Biomed. Microdev. 13 (2011), 463-473
  13. ^ ET Lagally et al., J. Phys. D 37 (2004), R245-R261
  14. ↑ B. Bruijns et al., Biosensor-Basel 6 (2016), No 41
  15. ↑ TH Kim et al, Anal. Chem. 86: 3841-3848 (2014)