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Portable POC Device Simultaneously Detects SARS-CoV-2 RNA and Antibodies in Saliva

By LabMedica International staff writers
Posted on 09 Aug 2022
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Image: The eRapid Sensor for COVID-19 (Photo courtesy of Harvard University)
Image: The eRapid Sensor for COVID-19 (Photo courtesy of Harvard University)

As the COVID-19 pandemic runs its course, the questions we ask ourselves have evolved, from how do I know if I’m infected to how strong is my immunity to which strain of the virus do I have. As new variants continue to emerge, it’s likely that we’ll keep asking ourselves those questions, often at the same time. Now, a new diagnostic device in development could offer a way to get answers to all of them in a couple of hours without needing to send samples to a lab.

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University (Cambridge, MA, USA) have created a prototype point-of-care device that combines the institute’s eRapid and SHERLOCK technology from Sherlock Biosciences (Cambridge, MA, USA) into a single, postcard-sized system that can simultaneously detect the presence of both SARS-CoV-2 RNA and antibodies against the virus in a patient’s saliva, potentially along with multiple other biomarkers. As a prototype, the device is still a preliminary model and is not yet ready for large-scale manufacturing and distribution. So far, however, signs are promising.

The microfluidic system consists of multiple reservoirs, channels and heating elements to automatically mix and transfer substances within the prototype device without needing input from a user. In the first chamber, saliva is combined with an enzyme that breaks open any viruses’ outer envelopes to expose their RNA. Then the sample is pumped into a reaction chamber, where it is heated and mixed with loop-mediated isothermal amplification (LAMP) reagents that amplify the viral RNA. After 30 minutes of amplification, a mixture containing SHERLOCK reagents is added to the chamber, and then the sample is pumped onto an eRapid electrode.

In the absence of SARS-CoV-2 genetic material in the mixture, single-stranded (ssDNA) molecules with biotin attached to them bind to a molecule called peptide nucleic acid (PNA) on the electrode’s surface. The biotin then binds to another molecule in the mixture called poly-HRP-streptavidin, which causes a third molecule, tetramethylbenzidine (TMB) to precipitate out of the liquid solution as a solid. When the solid TMB lands on the electrode, it changes its electrical conductivity. This change is detected as a difference in the amount of electrical current flowing through the electrode, indicating that the sample is free of the virus. If any SARS-CoV-2 genetic material is present in the saliva sample, however, the CRISPR enzyme within the SHERLOCK mixture cuts it as well as the ssDNA. This cutting action separates the biotin molecule from the ssDNA, so that when the ssDNA binds to PNA, it does not trigger the series of reactions that causes the TMB to precipitate onto the electrode. Therefore, the conductivity of the electrode is unchanged, indicating a positive test result.

In parallel, the team customized the remaining three eRapid electrodes by studding them with different COVID-related antigens against which patients can develop antibodies: the S1 subunit of the Spike protein (S1), the ribosomal binding domain within that subunit (S1-RBD), and the N protein, which is present in most coronaviruses (N). If a patient’s saliva sample contains one or more of these antibodies, they bind to their partner antigens on the electrodes. A secondary antibody that is attached to biotin will then bind to the target antibody, triggering the same poly-HRP-streptavidin/TMB reaction and causing a change in the electrode’s conductivity. The researchers tested these antibody-specific sensors using samples of human plasma from patients who had previously tested positive for SARS-CoV-2. The system was able to distinguish between antibodies against S1, S1-RBD, and N with over 95% accuracy.

Finally, the team tested the combined viral RNA and antibody electrodes using saliva from SARS-CoV-2 patients. They split the saliva into two portions, adding one portion to the antibody reservoir and the second portion to the RNA reservoir of the device. After two hours, they measured the electrodes’ readouts to see if they had correctly registered the presence of the antibodies and RNA. The team found that the multiplexed chips correctly identified positive and negative RNA and antibody samples with 100% accuracy, at the same time. It was also ultra-sensitive, able to detect the presence of RNA down to 0.8 copies per microliter. The prototype device’s low cost and compact design is user-friendly and minimizes the number of steps a patient needs to perform, reducing the possibility of user error. Customized cartridges could be easily manufactured to detect antigens and antibodies from different diseases, and could be fit into a reusable housing and readout device that a user would keep in their home.

“This diagnostic can enable cheaper, multiplexed monitoring of infection and immunity in populations over time, at levels of accuracy that are comparable to expensive lab tests,” said co-first author Devora Najjar, a graduate student at the MIT Media Lab and the Wyss Institute. “Such an approach could dramatically improve the global response to future pandemics, and also provide insight into which treatment individuals should receive.”

“Being able to easily distinguish between different types of antibodies is hugely beneficial for determining whether patients’ immunity is due to vaccines versus infection, and tracking the strength of those different immunity levels over time,” said Sanjay Sharma Timilsina, Ph.D., a former Postdoctoral Fellow at the Wyss Institute who is now a Lead Scientist at Stata DX. “Integrating that with viral RNA detection in a portable, multiplexed diagnostic platform provides a comprehensive view of a patient’s health both during and after an infection, which is essential for implementing public policy and vaccination strategies.”

“Currently, there is a lack of low-cost diagnostic platforms that can enable accurate detection of multiple classes of molecules without requiring a trip to a lab. Our system offers the best of both worlds – high accuracy and low cost in a multiplexed platform – and could provide a lot of value to both patients and clinicians at the point of care. Plus, it’s easily adaptable to a wide range of applications,” said Wyss Senior Staff Scientist Pawan Jolly, Ph.D.

Related Links:
Harvard University 
Sherlock Biosciences 

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