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Nanopore-Based Single-Molecule Detection Tech Shows Promise for Viral Load Tracking


NEW YORK – A label-free, non-amplification biosensor technology for detecting single nucleic acid molecules is at least equivalent to RT-qPCR for tracking fluctuating viral load levels in biofluids, making it potentially attractive for use in low-cost molecular diagnostics.

Comprising microbead-based target capture, optical trapping, and nanopore detection, the technology has been licensed by a subsidiary of Fujirebio and is also being developed for research use at the University of California, Santa Cruz's Center for Live Cell Genomics.

Scientists at UCSC, Brigham Young University, and the Texas Biomedical Research Institute demonstrated attomolar limits of detection for the system in research published in the Proceedings of the National Academy of Sciences last week, showing it could be used to track the four-week evolution of Zika virus and SARS-CoV-2 virus infections in six different types of body fluids from two primate animal models.

The primate models were developed in BSL-4 facilities at TBRI. With tiny marmosets, the group examined the rise and fall of Zika virus levels during a 28-day infection course using whole blood, urine, and semen samples. The work was supported in part by funding initially provided by the National Institute of Health's National Institute of Biomedical Imaging and Bioengineering in 2019.

In parallel, SARS-CoV-2 infections were studied longitudinally in baboons using nasopharyngeal throat swabs, rectal swabs, and bronchoalveolar lavage samples.

The biosensor chips themselves are the handiwork of the BYU team. The microscopic devices are microfabricated to combine fluidic channels with tiny channels to let light in, also called optical waveguides, and covered with a 300-nm-thick silicon dioxide sheet containing a laser-milled 20-nm nanopore.

In the PNAS study, after preparing the assorted primate biofluid samples the team captured target viral RNA using biotin-conjugated sequences and streptavidin-coated magnetic microbeads with a diameter of 1 micron.

The magnetic microbeads were then strategically trapped by beams of light, guided to the nanopore-region of the chip, and gently heated to release the nucleic acids in a process dubbed trapping-assisted capture rate enhancement, or TACRE.

A single molecule passing through the nanopore resulted in a blip of current, and the blips could be easily counted and used to calculate the amount of virus in the original sample.

Compared to RT-qPCR, the nanopore sensor and TACRE approach produced qualitative and quantitative agreement for all positive primate biofluid samples, the team noted.

The biosensor method also yielded viral load readings for samples that did not produce a RT-qPCR result, which the team concluded was due in part to the complexity of the PCR processing. The method also avoided the bottlenecks associated with qPCR, including reverse transcription, amplification, and standard curve calibration.

Overall, all the sample types were detected on the nanopore chip with a detection limit of 10 attomolar and a dynamic range of five orders of magnitude.

The team concluded that optical trapping-assisted microbead delivery and thermal release of target particles near the nanopore sensor "enabled rapid, high-throughput diagnosis by increasing the local target concentration up to over 1 million times," according to the study.

Commercial potential

The UCSC/BYU team has been developing the optofluidic nanopore technology for more than 15 years, but the genesis of the project dates to a casual conversation between UCSC colleagues Holger Schmidt and David Deamer about two decades ago.

Deamer was describing a method he co-invented using nanopores, and Schmidt, an electrical engineer specializing in optics, realized the approach could be incorporated into the optical devices he was developing with his BYU colleagues.

"I thought, it's such a beautiful, simple concept that has a lot of potential," Schmidt said in a recent interview. "It was one of the most impactful on-the-couch conversations I've ever had, for sure."

So, while Deamer was applying the principle to sequencing — and UCSC would eventually license the approach to Oxford Nanopore Technologies — Schmidt proposed using nanopores essentially as sensors.

He and his colleagues initially developed the method for protein sequencing, then began combining nanopores and optical detection to distinguish between two targets that were similar in either their electrical or optical properties. 

But the team kept encountering issues in "getting all these little molecules directly to the nanopore so they can be pulled through by an electric field," Schmidt said.

When a nanopore pulls a molecule through itself, the passage registers as a blip in the pore's electrical properties. But the tug of a nanopore's electric field on nearby molecules is related to proximity, such that distant ones do not experience enough force to be sucked in.

An expert in the use of optical trapping, also called optical tweezers, Schmidt knew that the piconewtons of force exerted by photons of light can be used to push and pull particles about. But extremely small particles also jiggle around randomly, governed by the properties of Brownian motion, so are challenging to trap with laser light.

In 2019, the team published proof that it could use an optofluidic method called anti-Brownian electrokinetic trapping to select, sort, and manipulate specific molecules in its chip.

Then, the challenge of delivering particles to the nanopore for detection was further overcome by using magnetic beads. "If we collect the molecular targets on a microbead, we can transport the bead directly to the nanopore using a light beam, and then basically park it there," Schmidt said. After that, "all the molecules are directly at the nanopore and can be detected very easily" once a slight rise in temperature releases them from the bead.

In 2020, the team described a method to detect viral hemorrhagic fevers using this approach, and in 2021, it showed the method could be used to detect SARS-CoV-2 viral antigens, albeit with spiked-in virus in a matrix rather than clinical samples.

The microbeads can also be easily functionalized to capture nucleic acids, proteins, or both in the same sample. Schmidt said his team is now developing its nanopore and optofluidic chip for multiplexed detection of different nucleic acid targets, such as SARS-CoV-2 and influenza, and to detect multiple types of analytes using the same microscopic device.

Multiplexed nucleic acid, protein, or multianalyte detection could be accomplished by spatial segregation, he said, performing capture of one target in one zone of the chip then shuttling sample to another zone for capture of the other target, for example. Microbeads from each zone could then be escorted to the nanopore separately and heated to release the targets. Alternatively, the chip could be designed to have two spatially segregated nanopores for detection of the different targets.

However, in cases where the current blips elicited — by, for example, target nucleic acids and proteins — are different enough, Schmidt said, capture beads could be mixed and delivered en masse to let the nanopore sort it out. The team has also developed a technique to increase the dynamic range of multiplexed target detection using fluorescent beads and a closed-feedback system that rapidly adjusts the power of the excitation laser. 

The ultimate cost of any diagnostic based on the optofluidic nanopore technology remains to be seen, but Schmidt said silicon is "pretty cheap," as are the semiconductor lasers used for optical trapping. The team is also working on integrating sample prep into the chip, which could potentially yield a sample-to-answer system that is "much less expensive than, say, a PCR machine," Schmidt said.

In the path to commercial application, Schmidt originally cofounded a startup called Fluxus in 2017 along with his BYU colleague, Aaron Hawkins.

Peter Wagner, the current president and CEO of Fluxus, confirmed in an email that the company has licensed the optofluidic and nanopore technology from UCSC.

Still, the technology "is at an early development stage," he said, so he could not provide details on the development activities, scope, timelines, or commercialization path at this time.

That said, the work of Schmidt and colleagues, including the newest iteration published in PNAS, is "extremely innovative," Wagner noted, adding that it "matches well with Fluxus' business model of bringing innovative detection technologies to market."

A potential accelerant for this business model may be found in the 2022 acquisition of Fluxus by Fujirebio for an undisclosed amount, as Fluxus is now a wholly owned subsidiary of the Japan-based global in vitro diagnostics firm.

"Fluxus is Fujirebio's Silicon Valley-based innovation subsidiary, with emphasis on detection technologies," Wagner explained.

Fujirebio is commercializing its Lumipulse fully automated chemiluminescent enzyme immunoassay system — tackling targets like procalcitonin, protein biomarkers of Alzheimer's disease, and neurodegenerative diseases like multiple sclerosis — and its acquisition announcement asserted that the Fluxus detection technologies would ultimately be used to enhance the Lumipulse system.

But, Fujirebio also offers contract development and manufacturing services to other global IVD companies, Wagner said, providing research, development, and manufacturing to support neurodegeneration, cancer, and infectious diseases diagnostics.

As part of this business, "Fluxus will also make its products and technologies available to IVD and life science companies as an integral part of Fujirebio's CDMO partnering strategy," he also said.

Whether the CDMO service will include the optofluidic nanopore tech is currently unknown, however. While Fluxus has licensed the technology and early development work is being done to address manufacturability and market readiness, "the outcome of that could also mean that the technology is not ready yet and hence will not be commercialized," Wagner said.

In the meantime, Schmidt is continuing to develop the optofluidic system, recently adapting the technique to a processing scheme involving a deep neural network for amplification-free, multiplexed, artificial intelligence-assisted pathogen detection on the fly.

And, he is also collaborating with other UCSC colleagues to bring the technology to the newly opened Center for Live Cell Genomics.

Supported by a $13.5 million NIH grant, the Center's other co-principal investigators, Sofie Salama and David Haussler, recently described a method to perform automated recording and imaging of the electrical activity of brain organoids. Schmidt and colleagues, meanwhile, described a method for live signal monitoring as well as an analysis algorithm to enhance single-particle sensors that produce large amounts of data over long periods of time.

The Live Cell team is now hoping to integrate the optofluidic nanopore chip into its cell-culture systems for brain stem-cell derived organoids, Schmidt said.

It expects to be able to monitor the molecules these mini-brains share with each other as they grow — such as exosomes, extracellular vesicles, circulating DNA, or microRNA — and to measure how the organoids respond to different stimuli and disease conditions.

In this sort of "point-of-incubator" manifestation, the optofluidic nanopore chip can analyze liquid drawn from the organoid incubator, "live and in real time, and all the time," Schmidt said, with researchers able to monitor the data remotely. Contrasted with the cumbersome process of performing individual PCR analyses, for example, Schmidt sees this approach as potentially saving time and money.