NEW YORK – A team of global researchers has developed a new method to create rapid, low-cost molecular diagnostic tests, using loop-mediated isothermal amplification (LAMP) and compaction oligos to generate balls of nucleic acids that can be easily detected with a biosensor.
Costing less than $5 per test, their technology is expected to be commercialized within the next few years.
In a study published last week in Science Advances, researchers from the Karolinska Institute, as well as from Rutgers University, Yale University, Stanford University, and Nanjing Agricultural University, showed that they could rapidly detect and quantify compacted balls of amplified DNA, or DNA nanoballs, using an impedance cytometer.
The published method deploys a standard LAMP reaction to amplify DNA then adds two compaction oligos that serve to snarl the DNA together into balls approximately 1 micron in diameter.
This use of nanoballs essentially "transforms the problem of detecting DNA from a nanometer scale to a micrometer scale, which significantly simplifies it," said Muhammad Tayyab, a graduate student at Rutgers University and corresponding author on the study.
As first described in 2009, DNA nanoballs are more typically used in sequencing technologies. They are an element of core proprietary Complete Genomics sequencing technology that was acquired in 2013 by MGI Tech and incorporated into its recently launched high throughput DNBSeq-T20x2 sequencer.
Compaction oligos have also been used in sequencing applications along with rolling circle amplification to enhance detection and were recently deployed in a study to enhance the sensitivity of a fluorescence-based point-of-care nucleic acid test for Candida albicans. However, the use of compaction oligos to create DNA nanoballs for biosensor detection is novel, and for the study, the researchers created the compaction oligos by inserting linker and spacer sequences in two LAMP primers against the Orf1ab region of the SARS-CoV-2 genome. They also developed compaction oligos for tuberculosis, HIV, influenza, and a beta-lactamase gene that confers antibiotic resistance using a similar approach and showed that these also form nanoballs.
Condensing amplified DNA into balls allowed the researchers to use simple detection techniques, as compared to standard fluorescent or colorimetric methods. This, in turn, eliminated much of the instrumentation typically used in diagnostics, Tayyab said.
"The size of these nanoballs is actually very comparable to the size of cells," he said, so the detection method of choice for the team is an electrical impedance cytometer. When a DNA nanoball flows between two electrodes, the resulting momentary spike in current detects the nanoball, and the number of spikes correlates to the amount of starting substrate. The team has also developed a custom machine-learning algorithm to quantize the spikes, Tayyab said.
Electrical impedance biosensors have been used to detect blood cells infected with malaria-causing parasites, for example, and for bladder cancer grading. Indeed, biosensors in general are increasingly being used in diagnostics, with companies like Aptitude Medical and DiagMetrics developing test devices incorporating this technology.
But while many biosensors need to be functionalized for diagnostic testing — essentially by coating them with special substances in order to detect pathogen protein or DNA — the nanoball method only requires the balls to flow between two electrodes in a microfluidic device.
In the Science Advances study, the team tested 15 clinical samples from patients with COVID-19 and showed the number of discreet DNA nanoballs was correlated to the Ct value obtained using PCR testing. Even samples in the range of Ct 26 to Ct 27, which presumably represented a low viral load in the infected patients, yielded close to 1,000 detection events.
The method can potentially be multiplexed by incorporating biotin or gold nanoparticles that change the dynamics of the spike detected by the biosensor, Tayyab said, and the current single-chamber microfluidic chip design could be expanded to have multiple chambers, which would also enable multiplexing.
Collaboration and commercial intentions
Mehdi Javanmard, an engineering professor at Rutgers University and coauthor on the work, said the team came together during the pandemic, inspired by a desire to create low-cost, highly accurate diagnostics for COVID-19.
"To be able to detect viruses when infected people are pre-symptomatic, you need tests that are more sensitive and can catch the infection at early stages when viral concentrations are really low," he said in an email.
Vicent Pelechano, another coauthor on the Science Advances study and a microbiology researcher at the Karolinska Institute, said the team's bi-weekly online discussions during the pandemic were what ultimately led to the new method.
"We realized that [Javanmard] had a smart way to detect DNA using electricity," Pelechano said, but that his team's original method was bead-based, which increased cost and hands-on time.
Pelechano's team of molecular biology experts then reasoned that instead of using traditional molecular biology reactions, it could invent a new one. "We found a very simple way to transform the product of a DNA amplification into small compact balls of DNA," he said. This approach yields what is essentially a "quanta" of DNA amplification "that our colleagues who are electric engineers can detect easily," he said.
Lars Steinmetz contributed to the work as well, along with alumni from both the Stanford Genome Technology Center, which Steinmetz co-directs, and colleagues in his group at the European Molecular Biology Laboratory.
Steinmetz — a professor of genetics at Stanford who cofounded Sophia Genetics and Recombia Biosciences — said his team at the SGTC began their part of the project independently looking at using microfluidics for analyzing drug-resistant bacteria, but when COVID hit, the team pivoted and brought its insights to the online collaborations.
"With this interdisciplinary combination of expertise on one Zoom call, the application of the nanoball technology was dreamed up — something that wouldn't have eventuated from just one of our groups alone," Steinmetz said in an email.
The estimated cost of the diagnostic test will be in the range of $5, which is significantly lower than most molecular diagnostic tests currently commercially available.
Steinmetz and Pelechano are also each cofounders of existing startups — Levitas Bio and 3N Bio, respectively — but they don't currently have any plans to commercialize the technology through these companies as the core technologies and aims of their firms are somewhat different.
The team does have a pending patent for the technology, according to Javanmard, and is continuing to develop it with the intention of performing larger clinical studies.
Now, "we're seeking academic funding and exploring commercial partnerships," he said, adding that the team anticipates "seeing this as a product on the market in three to five years."