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Dutch Researchers Craft Glow-in-the-Dark POC Infectious Disease Test


NEW YORK – Researchers from Eindhoven University of Technology in the Netherlands have developed an infectious disease molecular testing method that uses bioluminescent proteins that they hope to eventually apply at the point of care.

The technique, which utilizes recombinase polymerase amplification (RPA) and CRISPR-Cas9 enzymes to detect RNA and DNA in a variety of sample types, is described in a new paper published this week in ACS Central Science.

The researchers were driven by a desire to create a molecular testing alternative to PCR, which was critical during the SARS-CoV-2 pandemic but requires specific equipment and can take hours to return results, lead author Maarten Merkx, a professor of biomedical engineering at the Eindhoven University of Technology, said. The team wanted to develop something that was much faster than traditional PCR but still highly sensitive, and thus its luminescent nucleic acid sensor, or LUNAS, platform was born. 

The two-step method requires no instrumentation beyond a heater and digital camera and returns results in about 20 minutes, as both steps are performed at the same time in the same sample. A sample — in the case of the ACS Central Science paper, the researchers used nasopharyngeal swabs — is amplified using RPA, which requires a relatively low temperature of about 40 degrees Celsius. Merkx noted that the lower temperature was the main reason the team chose RPA over more common amplification methods, such as loop-mediated isothermal amplification. 

As the sample is amplified, reagents that were added for detection of the target are activated. Those reagents, which make up what the researchers call the "sensor," consist of two CRISPR-dCas9 proteins with guide RNAs designed to bind to specific sequences of SARS-CoV-2, as well as a substrate. Both proteins also contain bits of luciferase that, when bound to the target, form an active enzyme that reacts to the substrate in the reagent mix and generates blue light. 

While a variety of tests use fluorescence or other methods of detection, Merkx said that those methods, when used with RPA, can create light scattering effects that result in background noise. Bioluminescence doesn't have that issue, so all of the light emitted in the test is coming from the reaction itself, which makes it particularly effective in more complex samples such as blood, he added.

However, the blue light generated is sensitive to the concentration of substrate in a sample, temperature, and time. To mitigate the potential fluctuations in results, the researchers added a second enzyme that produces green light and is also responsive to temperature, substrate concentration, and time, but does not respond to DNA or RNA and thus compensates for everything that can affect the bioluminescence. Because the intensity of the luminescence decreases over time as the substrate runs out, having a calibrator that is also affected by substrate concentration ensures the result can be corrected for that variable and that the test isn't time sensitive. The ratio of blue light to green light is measured by a digital camera, and if the ratio passes the control level, the test is positive. 

In the ACS Central Science paper, the researchers validated the test with both RNA isolates and regular nasopharyngeal samples and found that in RNA isolate samples with low cycle threshold (Ct) values — indicating a high concentration of SARS-CoV-2 RNA — the test identified all positives correlated by RT-PCR. Sensitivity was slightly lower for non-extracted samples, the researchers noted. For those with higher Ct values, and likely lower concentrations of RNA, the test correctly identified 21 out of 25 positive swab samples and 43 out of 55 positive RNA isolate samples. For both types of samples, all true negatives were identified by the test, including those that were positive for other respiratory viruses. 

The researchers added that the sensitivity could likely be further improved by increasing the sample input volume, decreasing the volume of the swab collection medium, or by higher-fold concentration in an RNA extraction step. 

Merkx also noted that there is little sample handling involved beyond the addition of the reagents and the incubation, so professional expertise isn't required. That flexibility primes it for use in point-of-care settings, which is where the researchers are targeting, although there are still multiple steps before it's available. Right now, the test could be easily implemented in a laboratory setting, but for point of care, it would need to be integrated into a cartridge or device with a specific reader — steps that would be better taken by a company rather than a research team, Merkx said. 

The researchers have developed other bioluminescent assays and have a startup, Lumabs, to commercialize those, but the focus of that startup is on immunoassays rather than molecular tests. Because the use of bioluminescence for point-of-care testing is relatively new, there aren't a lot of big commercial players in the market, Merkx said, but the team is open to partnerships with any parties that want to commercialize the test.

Sherlock Biosciences has developed its INSPECTR (Internal Splint-Pairing Expression Cassette Translation Reaction) platform, a synthetic biology-based system that can be programmed to distinguish targets based on a single nucleotide at room temperature without an instrument through a bioluminescent signal, but there are few other companies utilizing bioluminescence for diagnostics. 

However, other researchers have utilized bioluminescence for nucleic acid detection, including a Chinese team with researchers from Peking University and the Chinese Academy of Sciences that developed a DNA detection system using dCas9 proteins linked to luciferase to detect tuberculosis. A group based out of the University of Geneva developed a luciferase-based method to detect microRNAs, described in a 2020 paper also published in ACS Central Science. 

Researchers from David Baker's lab at the University of Washington, meantime, published a paper in Nature last month explaining the development of new luciferases using deep learning methods that could have applications for medicine and biotechnology. 

Nicolas Winssinger, an author on the 2020 paper and a professor of organic chemistry at the University of Geneva who was not involved in the creation of the LUNAS test, said via email that the work of Merkx's team was "brilliant," noting that there are similar technologies but that it is "unique in providing real-time monitoring and yielding a ratiometric readout." That combo "gives it a robustness that is very appealing," he added, although it still needs to be clinically validated. 

The test can also be used beyond SARS-CoV-2 for any infectious disease or RNA or DNA detection application as long as the RPA primers and guide proteins are modified, Merkx said. While the team started with SARS-CoV-2 because it was "the obvious thing to do" during the pandemic, they are also exploring its use for sexually transmitted infections and envision it being used in a general practitioner's office for quick and accurate results, he noted. Merkx added that the first commercial application for the technology may not be SARS-CoV-2, as it would depend on the business strategy of whatever company may license the technology. But "since the platform is generic, many possible applications can be considered."