NEW YORK – Combining CRISPR and antibody testing, researchers at the Wyss Institute for Biologically Inspired Engineering have created an assay to detect both RNA from and antibodies to SARS-CoV-2 in one sample.
The team developed a lab-on-a-chip that can concurrently detect virus RNA and immunoglobulins in saliva within two hours. Describing the method in a paper published in Nature Biomedical Engineering last month, the researchers addressed how to integrate CRISPR-based nucleic acid sensing with an electrochemical platform that had been validated to detect antibodies in an attempt to provide answers about not only the presence of the disease but also the body's response to it, said Devora Najjar, a research assistant in bioengineering at the Massachusetts Institute of Technology and one of the authors on the paper.
Before the pandemic, it didn't feel necessary to combine antibody detection and RNA detection for diseases, she said. But "unfortunately, now we're all just very acutely aware" of questions surrounding our immune and antibody levels, so it "felt a lot more relevant, even if they aren't … usually tests that are integrated together," she added. "It just felt right in terms of trying to get a full picture of the disease state."
The team aimed to figure out how to combine the pieces of the puzzle to "design a microfluidic chip that can do all of the molecular steps necessary" for RNA detection via CRISPR and loop-mediated isothermal amplification (LAMP), as well as electrochemical antibody detection, she said.
"We realized that separately the antibody detection and the RNA detection, they worked really well and they were really cool — but something we didn't see available on the market that we thought would be exciting is a test that can maybe take as long as a traditional PCR but can just provide you with a lot more information," Najjar said. "If you're going to be getting the sample from somebody, how can we get the most out of it that we can?"
The eRapid electrochemical device itself is not new — it has been validated for other, non-COVID-19 diseases in the past, said Pawan Jolly, a senior staff scientist at the Wyss Institute and another author on the paper. The technology uses an antifouling nanocomposite coating that has antibodies or antigens attached to capture viral proteins, RNA, or antibodies, depending on the application.
For this COVID-19 test, the electrochemical sensor chip within the device contains four electrodes that are used to detect three SARS-CoV-2 antigens and a nucleic acid capture probe.
The test uses a saliva sample, with a small portion of the sample collected for the antibody detection and the rest put into the sample preparation chamber for the RNA detection process. On the RNA detection side, the sample combines high heat followed by an enzymatic denaturation step that inactivates nucleases and inhibitors from the saliva that could end up erroneously activating the reporter molecule in the CRISPR reaction, Najjar said.
Once that sample preparation step is completed, the sample is transported via microfluidics to a reaction chamber with a membrane acting as a capture platform for the nucleic acids present in the saliva, she said. There a LAMP reagent mix is added to the sample and incubated for 30 minutes. After that incubation, a CRISPR reagent mix is added to the sample and incubated for another 30 minutes, Najjar said.
After the incubation, the CRISPR-RNA and antibody samples are successively flowed over the electrochemical chip. If the CRISPR reporter molecules and COVID-19 antigens are present and bind to the chip, a signal is generated. That signal is measured via electrical current and fed into previously created calibration curves that provide the concentration of antibodies and a yes-or-no result for RNA detection, Jolly said.
The entire process takes about two hours but could be shortened if a couple of steps were optimized or the microfluidic pumping was faster, Najjar noted.
"There aren't that many assays that work across biomolecule type, which makes sense because the beauty of biology is that it can be so specific to targets," she said. "The idea of having this broader test feels a little bit more outside of the range of what people usually publish."
That range of results available from this test is a point of emphasis for the researchers, Najjar said. "If you're just looking at whether or not you test positive on a PCR [test], you're not getting the full picture of what your disease state and disease progression is."
As a result, "integrating antibody detection is actually a really great way of being able to understand on a much broader scale where your body's at and where your system is at in terms of whether or not you're infected or what stage of the infection or post-infection you are," she said.
Can Dincer, a junior research group leader at the University of Freiburg in Germany, is working with his team on a similar method to combine CRISPR-powered nucleic acid detection and antibiotic drug detection that was explained in a MedRxiv preprint from earlier this year. While their system doesn't require target amplification, unlike the Wyss device, and can detect six targets in 30 minutes, Dincer's method doesn't have integrated sample preparation, he noted via email.
He added that it's "great to have the simultaneous detection of the virus along with the immunity test" but said that the requirement of nucleic acid amplification, which needs different chemicals and reagents for each target beyond the reagents for the CRISPR reaction, makes the system "more complicated, expensive, … and slower."
Najjar noted that the device would be useful in a clinical or hospital setting since right now the electronic components "aren't at a price point" that would make them available in people's homes. But with "a bit of engineering and optimization," an at-home version could be developed that would have different cartridges or microfluidic chips for different assays that could be plugged into a single "multifunctional" device that would have the electrochemical components.
Jolly added that one of the team's goals was to create a test that can identify a patient's viral load and differentiate between whether a patient was previously infected with COVID-19 or vaccinated, which could make it useful for vaccine development in the future.
The underlying technology is also applicable across different disease and sample types, although a change in sample would require additional assay optimization, Najjar said.
Pathogens in which a user would be looking for DNA or RNA and an antibody response could all be detected with this method, and the next steps for the team are working with the technology to apply it to other pathogens.
Jolly added that the applications for other diseases depend on research grants but noted that the technology has seen a lot of interest from companies in the veterinary industry. He also said that the device could be used for multiplex tests, such as for sepsis relying on multiple different sepsis biomarkers.
Commercialization of the method depends on a variety of different players — while the Wyss Institute has patented this specific combination of the CRISPR and eRapid technology, Sherlock Biosciences is already commercializing the CRISPR tech, making any licensing agreements with outside companies tricky, Jolly said. However, there are different models available where the Wyss Institute could conduct sponsored research on a specific disease using this combination of technology for an outside company, or another firm could license eRapid and sub-license the CRISPR technology for their own test development.
In June, StataDx — cofounded by Jolly — exclusively licensed eRapid for use with neurological, cardiovascular, and renal diseases. And in 2020, Australia's iQ Group Global licensed eRapid for COVID-19 applications, intending to integrate the technology with iQ's transistor technology.