Multiplexed Electrochemical detects both Nucleic acids and Proteins

In this paper, we designed an electrochemical diagnostic device that bridges biology and electronics to simultaneously detect viral RNA and human antibodies for diagnosing and tracking the course of COVID-19 and potentially other infections.

Biological systems and electronic systems seem to be at odds with each other: biology is inherently “wet,” while moisture spells death for most cell phones and computers. Living organisms use the four-letter code of DNA, while electronics use the binary of ones and zeros. But in this paper, we figured out a way to bridge biology and electronics to create a new type of electrochemical diagnostic device that can detect both viral RNA and human antibodies at the same time, allowing us to diagnose and track the course of COVID-19 and potentially other infections.

The Wyss Institute for Biologically Inspired Engineering provides a unique collaborative environment where biotechnology researchers from different disciplines share ideas, spaces and equipment. This atmosphere fosters teams with diverse expertise to assemble and attempt challenging projects with high impact. The seed for our electrochemical diagnostic was born from one such collaboration between the labs of Wyss Core Faculty members Jim Collins, Ph.D. and Don Ingber, M.D., Ph.D., who is also the Institute’s Founding Director. The eRapid team, led by Ingber and Wyss Senior Staff Scientist Pawan Jolly, Ph.D., together with the SHERLOCK team led by Collins and Helena de Puig, Ph.D., a Wyss Postdoctoral Fellow, wanted to create a cheaper, more accessible alternative to the fluorescence readouts used in the first generation of CRISPR-based SHERLOCK diagnostics, which require costly instrumentation and infrastructure.

We realized that coupling biological sensing to electronic circuits would allow us to leverage signal processing and transmission elements to design sensors that assist with time- and location-sensitive data collection and disease tracking during potential outbreaks. 

In the search for a simplified sensor-based sample readout system, we centered on eRapid. eRapid is an electrochemical platform with rapid and low-cost anti-fouling surface chemistry that provides unprecedented sensitivity and selectivity, even in complex biological fluids including unprocessed whole blood, plasma, serum, and saliva. This unique, multiplexable technique provided the ideal framework to translate the molecular detection of a CRISPR-based system like SHERLOCK into an electronic sensor signal, and to create a super-sensitive diagnostic device that could detect molecules with lab-level precision in a non-lab setting. We started building this hybrid device and chose Lyme disease as our target application. Within a few months, we had gotten it to work. 

Then, the COVID-19 pandemic hit!  

We quickly noticed a common refrain in the news coverage about the lack of reliable diagnostic tests. We also recognized that most laboratories were working on either detecting viral RNA or antibodies against the virus, but not both. We were confident that we could successfully detect the presence of DNA and RNA molecules electrochemically thanks to our work on Lyme disease, so we chose to create an all-in-one SARS-CoV-2 sensor that could provide patients with a more complete picture of their disease state by detecting nucleic acids and antibodies at the same time. We chose saliva as our sample material because viral particles and antibodies can both be found there.

The greatest challenge we faced was designing a platform that could perform two separate and very different types of molecular reactions concurrently, then integrating them into one reporting system so that the results could be read simultaneously.  

Led by graduate student Devora Najjar with the help of Mohamed Yafia, we engineered a 3D-printed microfluidic system consisting of multiple reservoirs, channels, and heating elements to automatically mix and transfer substances within the prototype device without needing additional user input.

In the first chamber, saliva is heated and combined with an enzyme to extract the viral RNA and inactivate possible reaction inhibitors. Then the sample is pumped into a reaction chamber with a membrane that captures the viral RNA, where it is incubated with loop-mediated isothermal amplification (LAMP) reagents that amplify the viral RNA. After amplification, a mixture containing SHERLOCK reactions is pumped into the chamber, and then the sample is pumped onto an eRapid electrode that reports on the presence of SARS-CoV-2 viral RNA in the saliva.

While the device was being developed, Joshua Rainbow, who was a visiting graduate student from the University of Bath, UK, further developed the CRISPR electronic output by incorporating peptide nucleic acids (PNA) as a probe in place of DNA, thereby reducing the detection of reporter DNA time down to 5 min.

In parallel, Sanjay Sharma Timilsina, who was working as a Postdoctoral Fellow at Wyss Institute, led the customization of three other eRapid electrodes by conjugating 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). Several commercially available antigens were screened to develop the immunoassay with high sensitivity and specificity. If one or more of the target antibodies are present in a patient’s saliva, they will bind to their partner antigen, which will be detected by the eRapid electrode as a change in conductivity.

Finally, using the completed microfluidic device, we tested the combined viral RNA and antibody electrodes using saliva from SARS-CoV-2 patients. Our electrochemical platform can detect SARS-CoV-2 at accuracies comparable to or better than traditional laboratory-based techniques with the additional advantage that it provides an electronic readout using minimal instrumentation and patient sample, making it ideal for interfacing with electronic patient records and telemedicine.

Our device combines electrochemical readouts, microfluidics, novel bioengineered surfaces, and biotechnology advances to address the critical need for simultaneous SARS-CoV-2 viral RNA and serological detection in an integrated, low-cost and easy-to-use chip. 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 for clinical or even at-home use.

In addition, our platform can be used for longitudinal evaluation to titer antibodies in large populations to determine the strength and duration of antibody activity and to differentiate innate vs. vaccine-induced immunity. We expect that these new capabilities of interfacing viral RNA and host antibody detection can become a major tool in our arsenal for addressing future pandemics.  

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