GRAPHENE FIELD EFFECT BIOSENSOR FOR CONCURRENT AND SPECIFIC DETECTION OF SARS-COV-2 AND INFLUENZA

The SARS-CoV-2 pandemic has highlighted the need for devices capable of carrying out rapid differential detection of viruses that may manifest similar physiological symptoms yet demand tailored treatment plans. Seasonal influenza may be exacerbated by COVID-19 infections, increasing the burden on healthcare systems. In this work, we demonstrate a technology, based on liquid-gated graphene field-effect transistors, for rapid and ultraprecise detection and differentiation of influenza and SARS-CoV-2 surface protein. The device consists of 4 onboard graphene field-effect electrolyte-gated transistors arranged in a quadruple architecture, where each quarter is functionalized with either antigen-specific antibody or chemically passivated control. The antigen-antibody interaction is dependent on uniform diffusion of virus delivered in low ionic strength phosphate buffer solution, entailing a facile operating procedure, where the user adds a drop of the viral surface protein solution onto the device. Our sensor platform was tested against a range of concentrations of viral surface proteins from both viruses with the lowest tested and detected concentration at [~]50 ag/mL, or 88 zM for COVID-19 and 227 zM for Flu, 5-fold lower than the values reported previously on a similar platform. Unlike the contemporary standard, RT-PCR test, which have a turnaround time of a few hours, the reported graphene biosensor technology has a fast response time of [~]10 seconds enabling rapid diagnosis. Furthermore, the antibodies tested were confirmed to be antigen-specific through cross-reactivity tests. Thus, we have developed a multi-virus, highly sensitive and specific detection tool for rapid diagnostic applications for contemporary, emerging, and future viruses.

Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.