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Study Describes 'World's Smallest' qPCR Platform

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NEW YORK (GenomeWeb) – An international team of researchers has developed a real-time PCR platform they claim is the smallest fully autonomous qPCR system in the world.

The platform weighs 90 grams and measures about 10 cm long, 6 cm wide, and 3.3 cm tall. It uses a 12V battery for power, and can process two samples plus a positive and negative control using standard 40-cycle qPCR in less than 35 minutes. Captured data is stored on the device and can be exported via a USB port. The four samples are pipetted as 0.2-microliter droplets onto a disposable glass coverslip before being added to the system, but future devices may be simplified to become sample-to-answer.

The research team was made up of scientists at the Saarbrucken, Germany-based branch of the Korea Institute of Science and Technology — also called KIST-Europe — as well as Brno University of Technology in the Czech Republic, Northwestern Polytechnical University in Xi'an, China, and the Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST) in the US.

In a study published in Lab on a Chip last week, the researchers demonstrated the qPCR system amplified an amplicon from synthetic cDNA of the H7N9 influenza virus, and used serial dilutions to show that the system was capable of detecting as little as one copy of viral DNA with a mean efficiency in the range of 0.9 per cycle.

The researchers ultimately intend to develop a sample-to-answer system based on the device, project researcher Pavel Neužil told GenomeWeb.

The next step will be pre-clinical validation of the device with a transfected cell-based method, similar to one the group previously published in Angewandte Chemie International Edition, Neužil said. Following that, the team plans to conduct trials in collaboration with the Czech Armed Forces' Biological Defense Department at Techonin, as that agency can safely process a diversity of pathogens.

The group performed some of the device development at NIST labs as part of a long-term collaboration between Neužil and co-author B. Robert Ilic, the project leader of the CNST Nanofabrication Research Group. During a few weeks' visit to NIST, Neužil said the group was able to successfully fabricate devices for several projects.

"As a hands-on research-enabling facility, CNST offers a complete toolset, including lithography down to minimum feature sizes in the single-digit [nanometer] domain and above ... as well as a full complement of etchers, deposition tools, and countless state-of-the-art analysis tools," Neužil said. "The most valuable asset is the technical staff with a wealth of experience in fabrication and processing — they continuously push the limits, enabling new process flows, [and] are super friendly and helpful."

A prototype of the tiny qPCR device the group built was also used in work by Neužil and his colleagues which focused on a method to incorporate high-resolution melt data collection into the transition between extension and denaturation in each PCR cycle.

The present study, however, emphasized further miniaturization of the device, and some of the melt analysis precision was sacrificed. Specifically, the device uses standard temporal thermal cycling, as opposed to spatial thermal cycling in which the sample can move between heat elements. To accomplish this efficiently, the device relies on a "virtual reaction chamber," which is essentially a 0.2-microliter droplet of PCR reaction covered with mineral oil, Neužil said.

With minimal sample size, the device has minimal heat capacitance, so heating and cooling can also be rapid and not require extra power. The melt temperature was still used to confirm specific DNA was amplified, but was not suitable for HRM analysis.

However, multiplex detection on this device is now an option. The group's previously published system for PCR multiplexing, which uses a mixture of intercalating dye for one DNA and an FAM probe for a second DNA, can detect both using a single fluorescent channel, and "this can be performed by this small PCR as well," Neužil said.

Unlike other miniaturized PCR systems — such as Ubiquitome's Freedom4 qPCR device or Amplyus' miniPCR for standard PCR — this system is autonomous, with an integrated LCD display so it doesn't require a smartphone, CCD camera, or laptop to operate.

The group further miniaturized the device by redesigning the optics so that the four optical read-out systems are integrated together. Yet each reaction droplet has its own fluorescence monitoring system, with voltage as the read-out. The method is thereby immune to ambient light, and no light shielding box is needed.

"Cameras, due to the limited frame rate per second, cannot do this," Neužil said, adding that the detector has a much larger area than the pixels of a CCD or CMOS imager, "making our system much more sensitive."

"We envision that in the future all the chemicals will be lyophilized on the glass, and one will only dispense the pre-treated template containing DNA or RNA," he said. "Eventually we will develop an entire sample-to-answer system which will use 40 microliters sample volume [and] pre-concentration [of DNA or RNA] will be automatically operated by using paramagnetic particles."

The device has no moving parts, enhancing its robustness. It could be made to run faster by tweaking the PCR chemistries, and the group also has the option of making the device even tinier in the future.

"We are very confident the system can be made even smaller, as we are far from its limit of detection," Neužil noted.

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