ABSTRACT
The COVID-19 pandemic has caused tremendous damage to the world. In order to quickly and accurately diagnose the virus and contain the spread, there is a need for rapid, sensitive, accurate, and cost-effective SARS-CoV-2 biosensors. In this paper, we report on a novel biosensor based on angiotensin converting enzyme 2 (ACE-2)-conjugated vertically-oriented silicon nanowire (vSiNW) arrays that can detect the SARS-CoV-2 spike protein with high sensitivity and selectivity relative to negative controls. First, we demonstrate the efficacy of using ACE-2 receptor to detect the SARS-CoV-2 spike protein via a capture assay test, which confirms high specificity of ACE-2 against the mock protein, and high affinity between the spike and ACE-2. We then report on results for ACE-2-conjugated vSiNW arrays where the biosensor device architecture is based on a p-n junction transducer. We confirm via analytical modeling that the transduction mechanism of the biosensor involves induced surface charge depletion of the vSiNWs due to negative electrostatic surface potential induced by the spike protein after binding with ACE-2. This vSiNW surface charge modulation is measured via current-voltage characteristics of the functionalized biosensor. Calibrated concentration dependent electrical response of the vSiNW sensor confirms the limit-of-detection for virus spike concentration of 100 ng/ml (or 575 pM). The vSiNW sensor also exhibits highly specific response to the spike protein with respect to negative controls, offering a promising point-of-care detection method for SARS-CoV-2.
ABSTRACT
Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.