Recently, Dana Pfister Sullivan, a product manager at Canon BioMedical, sat down with Gert de Vos, the inventor of the NEXTGENPCR instrument and Director at Molecular Biology Systems, B.V. (MBS). Gert has master’s degrees in biology and physics from Leiden University in the Netherlands. After teaching physics in Curaçao, Gert returned to the Netherlands and embarked on a career as an entrepreneur and inventor in the life sciences.
DS: How did you come up with the idea for NEXTGENPCR?
Well, I was in a meeting focused on the detection of pathogens in milk. During the meeting, we were discussing gel electrophoresis and touched briefly on PCR. One person at the table mentioned PCR chips. I was aware of how PCR chips worked, specifically how the liquid or sample is pumped through different temperature zones, and also that they required nanoliter volumes rather than microliter volumes. I thought, wouldn’t it be great if we could do something similar — not only move the liquid around, but instead move the whole enclosure. That is when I got the idea — so then I went home, and we started on prototypes for what would become NEXTGENPCR. The first experiment we did was around ten minutes, and it worked brilliantly. I said, “Wow, we have something here!”
DS: How is NEXTGENPCR different than standard end-point PCR instruments?
In a typical PCR instrument there is an aluminum or silver block with cavities that holds the individual tubes of the PCR plate. These tubes are made of polypropylene, and they have a 200 to 300 micron wall to help hold their shape. The block is heated to the desired temperature, usually 95°C for denaturation, and then cooled down to the desired annealing temperature. What happens then is interesting; the cooling is done by Peltier elements. The moment you start to use a Peltier element for cooling, the other side gets hot; so, you have to cool a lot more than just the block. When the other side gets hot, the efficiency tumbles; so, while it is initially cooling at eight degrees per second, after a few seconds it slows down to two to three degrees per second. Therefore, it takes some time for the instrument to achieve the correct annealing temperature. Also, the sample inside the tube relies on convection to reach the correct temperature. Rather than use the common Peltier technology, NEXTGENPCR uses temperature zones that eliminate the time it takes to heat and cool during a three-step PCR.
DS: How does NEXTGENPCR achieve such fast PCR?
The technology differs from typical thermocyclers in two ways that enable fast PCR. One, the instrument has three temperature zones, one each for denaturing, annealing, and extension. Two, we decrease the thickness of the well so that the liquid can reach the needed temperature quicker. Our plates have polypropylene enclosures where the PCR mix has been added inside. These enclosures are squeezed between two blocks at the correct temperature. So, if the sample has to go from 95°C to 60°C, the plate moves from the 95°C temperature zone to the 60°C temperature zone. The instrument then presses the blocks together, slightly deforming the enclosures, which allows the PCR reagents to not only mix instantly but also come to the desired temperature, essentially removing the ramp time.
DS: What was important to you when you were designing NEXTGENPCR?
That is an excellent question. While developing the NEXTGENPCR instrument, I met an expert in the PCR market. I think he sold his first PCR instrument in 1984 or 1985. He told me this is potentially a market-disruptive technology, but not in its current shape. He noted the need to have a microplate format because then it will be compatible with all the current downstream applications. We started to work together because of what he said. We started making an instrument that accepts microplate formats. Our plate matches the rim of your typical microplate; in the middle there is a polypropylene sheet with either 96 or 384 wells. Our blocks are flat faced, so it does not matter what plate format you use in the instrument; there are no changes needed.
In addition to the plate, it was important to achieve high-speed PCR while also considering those things important to a user such as how much space the instrument takes on the bench, how much energy is used, and, in the end, how much the device will cost.
DS: How are the plates and consumables used with the NEXTGENPCR instrument different than standard 96- and 384-well plates?
The plates are the same size as a typical microplate because we use the same outside rim. They adhere to the standard defined by the Society for Laboratory Automation and Screening, so they fit in all robotics – both upstream and downstream. However, they differ in the center where we have inserted polypropylene film that has either 96 or 384 wells with a well thickness of 30 to 40 microns. The user’s current PCR mix is pipetted into the wells and then sealed with a heat sealer to close the wells.
DS: You mentioned that energy usage was an important factor in the design. How is the instrument able to save energy?
That is simple to explain — since the instrument has three temperature zones, we don’t need to change the temperature of the blocks. This means we isolate the blocks in the denaturing zone and the extension zone, and in the annealing zone we are able to cool at a certain rate so that the instrument is capable of touchdown and stepdown experiments. Because of this isolation, the instrument is running at 150 to 170 watts rather than 800 to 1200 watts that a standard cycler uses. Then, if you take into account that a PCR experiment lasts between five and 20 times shorter with NEXTGENPCR, you can imagine the amount of energy you use. It could be up to 100 times less compared to other PCR instruments.
This is only the first part of our interview with Gert. Come back next week for the conclusion of our interview
. In the meantime, download the application note that explains how a 100 bp fragment was amplified in less than 2 minutes using NEXTGENPCR.
DOWNLOAD APPLICATION NOTE