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Digital PCR: Old Technology Brought to Life on a Novel Affordable Automated Platform

Paul Hung, Ph.D., started out as an electrical engineer, but halfway through his graduate studies at the University of California, Berkeley (UC Berkeley), he switched to bioengineering. “I was fascinated by how many unsolved problems and unlimited possibilities there were in biology and hoped that my engineering expertise could help,” Hung recalls. As a serial entrepreneur, and now the CEO of COMBiNATi, Hung saw “huge potential in field of genomics” and earned the 2018 SLAS Innovation Award for his presentation, “Microfluidic Siphoning Array: A Novel Scalable Digital PCR Integrated Platform.”

Hung joined Professor Luke Lee's group to study bioengineering and work with microfluidic circuitry for cell biology-based applications. With practical skills in hand, just after graduating, Hung started a company, CellASIC, with another member of Lee’s laboratory. They obtained Small Business Innovation Research (SBIR) funds, got their microfluidic cell culture product to market and became self-sufficient in about six years. At that point, the business was acquired by Millipore, a collaborator, who still markets the CellASIC product. As part of the acquisition, Hung joined Millipore and learned how a large, global business scales up and extends the reach of a product.

Advances in genomics caught Hung’s eye, particularly applications of PCR technology, and eager to apply his new business knowledge to another start-up, Hung got in touch with some former colleagues and formed COMBiNATi in 2016. Co-founders Hung, Andrew Zayac, Megan Dueck, Ph.D., and Samuel Yang, M.D., each contribute a key piece of expertise to the team. “We are looking to bring an integrated digital PCR platform with injection molded microfluidic consumables from lab to marketplace to advance biomedical research…and to eventually deliver the best in class digital nucleic acid test platform enabling precision personalized medicine,” explains Hung. COMBiNATi was awarded two SBIR grants to develop an integrated digital PCR platform and micromolded siphoning array and to demonstrate the viability of the system. They now have a working prototype in hand.

As explained in his award-winning presentation at SLAS2018, key features of the COMBiNATi system include:

  • Digital PCR using open source chemistry
  • An injection molded microfluidic siphoning array with a semi-permeable membrane
  • Photographic imaging available throughout the process
  • Independent control of each module of the system

Why Digital PCR?

Referred to as “molecular photocopying” by the U.S. National Institutes of Health, PCR has revolutionized the study of DNA and evolved from a basic research tool to a highly sensitive diagnostic tool and more. Modifications to original techniques allow real-time quantification of amplified DNA, but while it is considered the gold standard, real-time or quantitative PCR (qPCR) has limitations. As Hung explains, “the results of bulk amplification of heterogeneous DNA fragments in a sample can be quantitated, but it requires the use of a standard curve and calculation of what was present in the sample. This is indirect and very imprecise for the detection of rare events.

“What’s exciting about digital PCR (dPCR) is that you are able to detect very rare events and directly count positive results, rather than calculating them. This increases precision,” explains Hung. He talks about how dPCR techniques were first published in the 1980s and 1990s but it has taken technology time to catch up. “The basic principal behind limiting dilution or digital PCR is that single or low numbers of nucleotide targets are partitioned before being amplified. After the amplification you count the number of partitions with a positive reaction to get your result,” Hung says, “Digital PCR has enabled liquid biopsies based on known genetic biomarkers present in the blood to be used in clinical medicine for early detection of cancer recurrence or infections and has potential in the realm of personalized medicine.”

A comprehensive review of dPCR, recently published in SLAS Technology, compares dPCR with qPCR and discusses different dPCR partitioning methods, statistics, commercially available systems and applications. The author concludes, “dPCR substantially improves precision in counting single molecules and resolving a small number of copies in the presence of inhibitors or wild-type populations. However, it does so at the expense of throughput.” There are several companies with products on the market using dPCR; COMBiNATi is coming at it from a different perspective.

Why Use COMBiNATi’s System?

Familiar Workflow and Chemistry

The COMBiNATi team has designed their system with the user in mind. Hung believes that “every scientist, every lab in this world needs access to this technology to make data better. It will be through the combined effort of all labs that we can truly advance science. We want to help by creating technology that is affordable and does not require new and complicated workflows.” To that end, their system provides an automated workflow, uses open chemistry, has real-time image acquisition with single color multiplexity and the injected molded device ensures consistent and robust quantified data.

Workflow has been designed to make the process equivalent to qPCR workflows already in use. Hung recalls this statement by one of his professors, “Biology is already complicated enough. Don’t make it more complicated. Don’t change our behavior.” With that in mind, “the automated workflow takes place on a single instrument that requires only one liquid transfer step – loading of the device. The device does not require any proprietary reagents. After loading the sample, the machine takes care of the rest – reagent partitioning, thermal cycling and image acquisition. With a simple, familiar workflow, you now have quantitative data with a lower detection limit and higher precision,” Hung explains. He believes the platform can be very affordable based on the discussion with reputable contract manufacturing partners.

Real-Time Imaging

“Unique to the COMBiNATi instrument is the single color multiplexity enabled by designing primers with different melt temperatures,” Hung explains. “Something that researchers love about real-time imaging is that they can do melt curve analysis, a capability of qPCR that none of the dPCR instruments in the market provide. Melt curve analysis after dPCR allows researchers to analyze genetic variations such as SNPs (single nucleotide polymorphisms), mutations and methylations with improved sensitivity and specificity. And, with the real-time imaging capability you can subtract background noise to minimize false positives by comparing images before and after thermal cycling.”

Consistent and Robust Data

The core of this instrument is the injection molded partitioning device that has a fixed number of partitions of known volume and batch-to-batch consistency. COMBiNATi has patented their microfluidic siphoning array technology in which bulk qPCR reagents are partitioned into discrete, separate chambers that they refer to as lollipops. “The prototype device has eight independent units, 5,000 chambers per unit, in a standard slide format (1 x 3 inches) that are connected by a serpentine loading channel,” Hung says “The device is first filled with the sample, then pressure is added to fill the lollipops and finally, gas or oil is flowed through the loading channel to isolate the chambers from one another. The concept is to mount 32 of these devices onto a standard microtiter plate format providing scalability and compatibility with robotics.”

The device is different from many lab-on-a-chip devices in a couple of important ways. “First, the device is not made from PDMS (polydimethylsiloxane) but from injected molded plastic,” Hung adds. “Injection molding technology today creates highly uniform and reproducible products in the hundred-nanometer range of precision (think DVD or Blu-ray discs). Right now, we can create lollipops with 40-micron diameter but expect to be able to go smaller and taller in a few years and to create even more complex designs. Second, instead of having to seal a PDMS device with glass, our device is gas impermeable until you add pressure. We have a semi-permeable thin film on the bottom of the device where outgassing occurs under pressure. This prevents fouling of the injection molded microfluidic devices by eliminating bubbles.” COMBiNATi plans to create application specific devices that vary in number and size of chambers that work with the modular instrument providing automation.

What about the Automation?

To keep things as simple as possible for the scientist, COMBiNATi has designed a machine that will handle all of the steps between sample preparation and data analysis and describes the processes. “The microfluidic siphoning arrays will be mounted on a machine that handles the sample partitioning, thermal cycling and image acquisition. The instrument has a device loading port, thermal cycler, pneumatic manifold that controls fluid actions and CMOS camera. It is controlled by software that provides independent control for key function – pneumatics, thermal cycling and image acquisition.”

Does it Work?

Hung and his team compared results to qPCR and to droplet dPCR (ddPCR) on the Bio-Rad system and found superior performance to qPCR and equivalent limit of detection and precision to ddPCR in an HIV long terminal repeats viral load assay and copy number variation analysis. They have also validated an Illumina next-generation sequencing library and, in a pathogen identification assay (using internal transcribed spacers), showed that the melt curve analysis can be used to identify and differentiate between two common pathogenic bacteria species.

What’s Next?

“We are now fundraising and looking to close our next round before mid-year 2018. The next step is to take the product to market. We have prototypes and have moved beyond proof of concept to being able to put someone else’s assay on the instrument and make it work,” explains Hung. “Additional funds will help us to bring the product to market but we also need partners. The greatest challenge is choosing the right partner or partners with whom to move forward. You need people who believe in you and want to take something new into the market with you. We see ourselves as a platform builder; we build instruments and the consumables but we don't provide reagents. We are very deliberately trying to leverage the open chemistry nature of the platform to enable us to partner with reagent providers in order to access the market faster.”

Appreciation for the SLAS Innovation Award

Hung felt his presentation was well received by the audience and said he had three groups approach him about potential collaborations. “I was surprised to win the award and very much appreciate it. The recognition is great and really helpful in legitimizing our technology. The technology does require some expertise to understand the basic principles of how we can accomplish this. There are important subtleties and it is not always easy to explain why it is interesting to someone without the right background. After all, digital PCR is not new and some people do not realize the potential of advancing an older technology in a new way,“ shares Hung.

Hung has been associated with SLAS for a number of years, dating back to before the merge of the Association for Laboratory Automation and the Society for Biomolecular Sciences in 2010. Sharing his perspective, Hung says, “I believe this organization is one of the best in bringing the engineering and technology-focused communities together. It is really helpful, especially for biologists to learn about new technologies and for the technologists or engineers to showcase their innovations to potential users.”

The Society recognizes one unique podium presentation at the SLAS International Conference and Exhibition each year for work that is “exceedingly innovative and contributes to the exploration of technologies in the laboratory, exceeds a benchmark or milestone in screening or the lead discovery process or demonstrates an advanced and integrated use of mature technologies.” The judging process is comprehensive and in the final step, judges attend all finalists’ presentations to score each for “impact on life sciences and technology, originality and creativity, quality of the science and the oral presentation.”

Plan now to opt in for 2019 SLAS Innovation Award consideration when the SLAS2019 call for abstracts opens in May 2018.

April 23, 2018