New portable diagnostic detects SARS-CoV-2 RN

As the COVID-19 pandemic has come full circle, the question we have been asking ourselves has evolved from “How can I know if I am infected?” “How strong is my immunity?” “What strain of virus do I have?” And as new variants continue to emerge, we may continue to ask ourselves these questions, often at the same time.

Now there is a way to get all the answers in a few hours without sending samples to the lab.A new point-of-care diagnostic device created by members of Harvard University’s Wyss Institute for Biotechnology e-rapid When Sherlock It has the potential to detect multiple other biomarkers in a single postcard-sized system that can simultaneously detect the presence of both SARS-CoV-2 RNA and antibodies to the virus in patient saliva.

“This diagnostic method allows for cheaper, multiplex monitoring of population infection and immunity over time, with a level of accuracy comparable to costly laboratory tests.” Such an approach could dramatically improve the global response to future pandemics and provide insight into the treatment individuals should receive.”

For prototype devices, Nature Biomedical Engineering.

Novel chemistry for novel viruses

The diagnosis is the result of an inter-laboratory collaboration of Wyss Core Faculty members. Jim CollinsPhD, and Don Ingber, MD, Ph.D., and the founding director of the Institute.of e-rapid Team led by Ingber and Wyss Senior Staff Scientist pawan jollyPhD, together Sherlock Team led by Collins Helena de PuigWyss Postdoctoral Fellow, Ph.D., recognized that while SHERLOCK-based diagnostics can detect molecules with great sensitivity, they are inherently limited by fluorescence-based readout systems. Like SHERLOCK, if he can figure out a way to convert the molecular detection of CRISPR-based systems into electrochemical signals like the one produced by eRapid, it could be used in non-laboratory settings for diagnostics with laboratory-level precision. law may be created. They started building this hybrid device and chose Lyme disease as the target application. Within months, they had it working.

Then came the COVID-19 pandemic.

“In the early days, everyone was working on a diagnostic that could detect either the SARS-CoV-2 virus or antibodies to it, but not both. It was known that the presence of DNA and RNA molecules could be detected electrochemically, thanks to , and multiplexed it with antibody detection to create an all-in-one test that could help track infections and fight pandemics. We decided to find a way to do that,” said de Puig, co-first author of the paper.

However, it has been difficult to create a platform that can integrate the detection of viral RNA and human proteins. The team needed to find a way to run two separate, but very different types of molecular reactions simultaneously, and integrate them into one of her reporting systems so that the results could be read simultaneously.

They chose saliva as the sample material because both virus particles and antibodies are there. His SHERLOCK portion of diagnostics, which detects the presence of SARS-CoV-2 RNA, required a device that could extract, concentrate, amplify viral RNA from saliva samples, mix it with CRISPR reagents, and deliver the results. Add the solution to the eRapid chip portion for detection.

The team designed a microfluidic system consisting of multiple reservoirs, channels and heating elements to automatically mix and transfer substances within a prototype device without requiring input from the user. . In the first chamber, saliva combines with enzymes to break the virus’ outer envelope and expose the RNA. The sample is then pumped into the reaction chamber where it is heated and mixed with loop-mediated isothermal amplification (LAMP) reagents that amplify viral RNA. After 30 minutes of amplification, the mixture containing the SHERLOCK reagent is added to the chamber and the sample is delivered to the eRapid electrode.

In the absence of SARS-CoV-2 genetic material in the mixture, biotin-conjugated single-stranded (ssDNA) molecules bind to molecules called peptide nucleic acids (PNAs) on the electrode surface. Biotin then binds to another molecule in the mixture called poly-HRP-streptavidin, which causes a third molecule, tetramethylbenzidine (TMB), to precipitate as a solid from liquid solution. When solid TMB adheres to the electrode, the electrical conductivity changes. This change, detected as a difference in the amount of current flowing through the electrodes, indicates that the sample is free of virus.

However, if SARS-CoV-2 genetic material is present in the saliva sample, the CRISPR enzyme within the SHERLOCK mixture will cut it as well as ssDNA. This cleaving action separates the biotin molecule from the ssDNA so that when ssDNA binds to PNA, it does not trigger a series of reactions that cause TMB to precipitate onto the electrode. Therefore, the conductivity of the electrode does not change, indicating a positive test result.

“By integrating the PNA-based assay with the poly-HRP-streptavidin/TMB reaction chemistry we created for this device, we were able to detect the presence of SARS-CoV-2 with four times greater sensitivity than the original fluorescence-based Sherlock. Co-first author Dr Joshua Rainbow, a former visiting graduate student at the Wyss Institute and now a PhD student at the University of Bath, says: “We are 100% sure of the presence of viral RNA. We were able to detect it with an accuracy of

greater than the sum of its parts

In parallel, the team customized the remaining three eRapid electrodes by studding them with different COVID-associated antigens that allow patients to generate antibodies: the S1 subunit (S1) of the Spike protein, ribosomes within that subunit. binding domain (S1-RBD), and the N protein (N) present in most coronaviruses. If a patient’s saliva sample contains one or more of these antibodies, these antibodies will bind to partner antigens on the electrode. A secondary antibody attached to biotin binds to the target antibody and triggers the same poly-HRP-streptavidin/TMB reaction, changing the conductivity of the electrode.

The researchers tested these antibody-specific sensors using samples of human plasma taken from patients who had previously tested positive for SARS-CoV-2. The system was able to distinguish antibodies against S1, S1-RBD, and N with greater than 95% accuracy.

“The ability to easily distinguish between different types of antibodies is invaluable in determining whether a patient’s immunity is due to vaccine or infection, and to track the strength of these different levels of immunity over time.” Dr. Sanjay Sharma Timilsina said.He is a former post-doctoral fellow at the Wyss Institute and is now chief scientist. StataDX“The integration of viral RNA detection into an ambulatory, multiplexed diagnostic platform will provide a comprehensive view of patient health during and after infection. Critical to implementation.” StataDX Commercialization of eRapid For neurological, cardiovascular, and renal applications.

Finally, the team used SARS-CoV-2 patient saliva to test electrodes combining viral RNA and antibodies. They split her saliva into two parts, adding one part to the antibody reservoir and the second part to the device’s RNA reservoir. After 2 hours, the electrode readings were taken to check whether the presence of antibody and RNA was correctly recorded.

The research team found that the multiplexed chip accurately identified positive and negative RNA and antibody samples simultaneously with 100% accuracy. It was also ultrasensitive to detect the presence of RNA down to 0.8 copies per microliter.

“Currently, there is a lack of low-cost diagnostic platforms that can accurately detect multiple classes of molecules without going to the lab. Our system offers both high accuracy and low cost in a multiplexed platform. It offers the best of both worlds and can deliver a lot of value to both patients and clinicians at the point of care.

The low-cost, compact design of the prototype device is easy to use, minimizes the number of steps the patient needs to perform, and reduces the potential for user error. Customized cartridges can be easily manufactured to detect various disease antigens and antibodies and can be attached to reusable housings and readers that users keep at home.

“What excites me about this diagnostic device is that it combines a flexible design with a high level of accuracy that could become a major tool in our arsenal to deal with future pandemics. said Collins, co-lead author and Professor Thermere. She holds a PhD in Medical Engineering and Science from the Massachusetts Institute of Technology.Additionally, Collins is the co-founder of Sherlock Biosciencesis developing the Wyss Institute’s SHERLOCK technology into the diagnosis of COVID-19 and other diseases.[BL1]

“I congratulate these teams who have come together amidst a global crisis that has shut down most activities to create new and useful things that bring great promise to point-of-care diagnostics and the management of a wide range of diseases around the world. I am very proud of the world,” and co-lead author, Judah Folkman Professor of Vascular Biology Harvard Medical School and Boston Children’s Hospital, and Hansjörg Wyss Professor of Biomedical Engineering at the Harvard John A. Paulson School of Engineering and Applied Science.

Other authors on this paper include Mohamed Yafia, Nolan Durr, and Hani Sallum of the Wyss Institute. Galit Alter at MGH, MIT, and the Lagon Institute at Harvard. Jonathan Li of Brigham and Women’s Hospital (BWH), Xu Yu of Ragon Institute and BWH, and David Walt of Wyss Institute, HMS and BWH. Joseph Paradiso of the MIT Media Lab and Pedro Estrela of the University of Bath.

This work was supported by the Wyss Institute for Biotechnology, Harvard University, the Paul G. Allen Frontiers Group, the Natural Environment Research Council (NERC) GW4 FRESH CDT, Fonds de recherche du Québec nature et technologie, and Ms. Enid . Schwartz, Mark and Lisa Schwartz Foundation, Massachusetts Consortium for Pathogen Readiness, Ragon Institute, Harvard University Center for AIDS Research.