Product Survey: Thermocyclers

Alternative Heat Production
by Harald Zähringer, Labtimes 03/2016

What have hot waterbaths, Peltier elements, hot air, electric resistance, magnetic induction, Ranque-Hilsch vortex tubes, infrared light, lasers and photons in common? They may all be used as heat sources in thermocyclers.

It’s been three years since our last product survey on thermocyclers (see Lab Times 4/2013, p. 47), so what has happened since in the world of PCR machines? Well, it is still dominated by Peltier heated block cyclers but alternative heating concepts are gradually gaining ground. And there is another trend that cannot be overlooked: the number of small, easy-to-use, portable, pocket-sized thermocyclers suitable for field studies or point-of-care diagnostics is growing rapidly.

Garden variety block cyclers are equipped with Peltier heated aluminium or silver blocks, delivering heating rates of about 2 to 5°C per second and slightly lower cooling rates. Coating the silver block with a thin gold layer considerably enhances ramp rates, especially in combination with thin wall propylene tubes, which mould into the block holes like a second skin. High end block cyclers based on this concept, such as Analytik Jena’s SpeedCycler2, may obtain ramp rates of 15°C/s, which are amongst the fastest of current Peltier heated block cyclers.

But even the most sophisticated heat transfer techniques cannot hide a major drawback, which has haunted block cyclers since their inception in the late nineteen-eighties: due to material inconsistencies and edge effects, the temperature is not evenly distributed throughout the heating block. Typical deviations from perfect block uniformity range from 0.3 to 0.5°C at PCR-relevant temperatures of 72° and 95°C. That may not sound too bad but in combination with other common inaccuracies of block cyclers, such as over- and undershooting temperatures, even slight variations in block uniformity may contribute to significant errors in PCR experiments.

Block uniformity has been considerably improved by a technique originally developed in Axel Scherer’s lab at the California Institute of Technology in 2006. His group came up with the idea to hollow out the silver block and fill the cavern with a thermally conductive liquid that is pumped through the hollow block during PCR cycling. The rapidly circulating fluid transfers the heat generated by the Peltier elements evenly across the block, providing a block uniformity of 0.1°C.

Hollow block cycler odyssey

The hollow block cycler has gone through quite an odyssey since Scherer’s first attempts to commercialise it via the start-up company Helios, in 2007. Helios was acquired by NGS giant ­Illumina in 2010, who initially sold the hollow cycler as the Eco Real Time PCR system but quickly lost interest in the ­thermocycler market and discontinued the Eco cycler in 2013. The NGS company sold the intellectual property rights for the Eco cycler to the British PCR distributer, PCRmax, who was purchased by the life science supplier, Bibby Scientific, in 2014. Shortly after the acquisition, Bibby Scientific relaunched the hollow cycler under the brand names Eco 48 (PCRmax) and Prime Pro 48 (Techne).

Scherer’s concept to enhance temperature uniformity with a fluid filled heating block is pretty smart. The easiest way to eliminate erratic temperature deviations in thermocyclers is, however, to completely abandon Peltier heated aluminium or silver blocks and switch to alternative heating techniques. One of the first implementations of this idea was an air-heated carrousel qPCR cycler, introduced by the small US biotechnology company Idaho Technologies in 1997, under the brand name LightCycler. The heating concept of the LightCycler is both simple and elegant: the qPCR reactions are carried out in thin, glass capillaries inserted into the holes of a rotor, spinning in an air-heated chamber. A small fan at the bottom of the chamber draws ambient air into the compartment that passes an electronically-regulated heating coil, placed in the inlet channel on top of the chamber. After circulating around the glass capillaries, the air leaves the chamber via laterally oriented outlet channels. To ensure fast cooling and heating rates, the fan rotates at lower speeds at the heating phases of the PCR cycles and accelerates during cooling. Detection of the amplicons is achieved by a laser beam focussed on the tip of the respective glass capillary in the measuring cell. To this end, a stepper-motor gradually rotates the carrousel to put the capillaries stepwise in the optical axis of the laser beam.

Rotary cycler with a twist

The then revolutionary heating concept of the LightCycler instrument quickly sparked the interest of ­Boehringer ­Mannheim. The German pharmaceutical corporation acquired the property rights for the technology, shortly before it was taken over by Swiss pharmaceutical giant Hoffmann-LaRoche, now Roche, in 1997. A few years later, John Corbett Senior, an engineer and inventor from Down Under, presented a similar rotary cycler system – but with a few little twists. Corbett’s rotary cycler, called Rotor-Gene, is based on a shallow centrifuge rotor, perforated like Emmental cheese, to allow maximal air circulation. The PCR is accomplished in standard, thin wall tubes looming out of the rotor places into an air-heated chamber. Heating and cooling of the chamber is achieved similar to the LightCycler by ambient air, forced into the compartment by a fan. But the detection of the PCR products is done slightly different. PCR tubes spin continuously and pass the excitation optics every 150 milliseconds to deliver a fluorescence signal that is detected by a photomultiplier tube. Rotary cyclers offer very fast ramp rates of up to 20°C/s but what makes them really stand out are untouched temperature uniformities of less than 0.02°C, e.g. for the Rotor-Gene.

Similar to the managers of Idaho Technology, John Corbett Sr converted his air-heated rotary cycler into money and sold his company Corbett Life Sciences together with the Rotor-Gene to Qiagen in 2008. But Corbett obviously didn’t want to rest on the 70 million dollars that he cashed in with the Qiagen deal. Together with his son, John Corbett Junior, he launched a new qPCR thermocycler, based on magnet induction heating, at the Biotechnica trade show in October 2015. As Corbett Sr told a television team filming his new Magnetic Induction Cycler (MIC) at the booth of his newly established company Bio Molecular Systems, he got the idea for his invention while heating coffee water on a magnetic induction cooktop. The underlying concept of the MI-Cycler is very straightforward: The Corbetts’ still rely on the tried and true rotary system of the Rotor-Gene and the fan for cooling but have substituted the heating coil with an electromagnetic coil that surrounds the aluminium rotor of the MIC. An alternating current flowing through the coil induces an alternating magnetic field around the coil that pervades the electrically conducting aluminium rotor and induces Eddy currents inside the metal. Due to the electric resistance of the metal, the induced Eddy currents instantly heat up the rotor.

Inspired by induction plates

Similar to classical rotary cyclers, the MI-Cycler shines with a very accurate well-to-well temperature uniformity of 0.05°C. However, heating rates of 4°C/s and cooling rates of 3°C/s are merely on average and provide qPCR cycling times of about 25 minutes. The explanation is simple: the rotating aluminium rotor acts similar to a heating block that transfers heat via conduction to the tubes. It is also possible to directly heat metal (iron) dotted PCR tubes through magnetic induction, which would lead to considerably faster ramp rates. According to John Corbett Jr, his company is already working on this technique to cut qPCR cycling times below ten minutes.

Other alternative heating systems already implemented in commercial PCR and qPCR cyclers are based on resistive heating of small disposable plates or silicon wafers. Examples are BJS Biotechnologies Xxpress Cycler and Cepheids Smart Cycler (see Lab Times 4/2013). But researchers are continuously tinkering on even smaller and especially more direct heating strategies. One is Laser PCR, developed by the German start-up company GNA Biosolution. The concept of Laser PCR is simple: instead of transferring heat from the reaction tube to the molecules of the PCR reaction mix, heat is directly produced inside the mix by focussing a laser beam on nanoparticles decorated with DNA templates functioning as tiny PCR platforms. After elongation of PCR primers binding to the DNA templates, a laser pulse heats up the nanoparticles to denature the newly synthesised double strands. GNA Biosolutions Laser PCR instrument Pharos is already in the test phase and may be applied for rapid diagnostics of pathogens.

Hot electrons

Photonic PCR, recently proposed by the group of Luke Lee from the University of California in Berkeley is also based on a new heating concept. (Son et al., Light: Science & Applications 2015, 4, e280). The photonic PCR cycler utilises LED light to heat a thin gold film in no time at all. As soon as the photons hit the gold surface, a plasmon-assisted light absorption occurs, which in turn excites surface electrons to higher energy states. The excited hot electrons reach temperatures of several thousand Kelvin within 100 femtoseconds – but they also cool down in a few pico seconds, if the light is turned off. Son et al. applied this technique to a photonic PCR thermocycler prototype, realising heating rates of approx. 13°C and 7°C, respectively. Needless to say that their ultimate goal is to turn the photonic PCR prototype into a cheap, small, portable thermocycler for molecular diagnostics – that’s where the money is.

First published in Labtimes 03/2016. We give no guarantee and assume no liability for article and PDF-download.

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