Title: Novel application of fluorescent nanodiamond (FNDP) for infectious viral antigens: A rapid detection test for ebola virus glycoprotein

Abstract

Background and objectives: There is dire need for rapid diagnostic tests of high sensitivity, efficiencyand point-of-test reporting capability to mitigate lethal viral epidemic outbreaks. Our objective was to replace the contemporary colorimetric (visual) LFA (Lateral Flow Assay) for Ebola virus (EBV) by a new operating system that operate by Near Infra-Red (NIR) emitted from Fluorescent Nanodiamonds-NV-z~200nm (FNDP-NV-200nm)deposited on test line within the lateral flow assay (LFA) format. The objective was to gain unprecedented superior sensitivity, speed, quantitativedata, and high throughput capacity. Specifically, we aimed to detail technical issues and feasibility of deploying the new near infra-red operating system (OS) based on FNDP-NV-200nm. Methods: FNDP-NV-200nm, 400nm or 800nm were linked to non-redundant anti-EBOV glycoprotein(GP) monoclonal antibodies (mAb) and tested for LFA performance by monitoring NIRemissions using an in vivo imaging system (IVIS) or an Opto-Electronic device (OED). Anti-EBOV glycoprotein (GP) humanized monoclonal antibody (mAb) c13C6 was linked to FNDP-NV-200nm forthe mobile phaseand a second, non-redundant anti-GP mouse mAb, 6D8, was printed on NCM at the Test-line. Goat anti-human IgG (GAH-IgG) served as a nonspecific antibody for capture of conjugatedFNDP-NV-200nm at the control line. Particles migration across the NCM in response to a fluidic pulse were assessed by NIR monitoring by an in vivo imaging system (IVIS). Results: FNDP-NV-200nm-c13C6 specifically and dose-dependently bound to recombinant EBOV GP in vitro and was effectively captured in a sandwich configuration at the Test line by mAb 6D8. FNDP-NV-200nm-c13C6 was captured on the control line by GAH-IgG. The OED quantitative analysis of NIR (obtained in less than 1 minute) was further validated byIVIS. Conclusion: FNDP-NV-200nm performance as a reporter for EBOV GP rapid diagnostic tests suggested an opportunity to replace contemporary colorimetric visual tests for EBOV GP and likely otherhighly lethal viral pathogens. Mobile, battery-operated OED adds portability, “Point of Need” (PoN) utility along with unprecedented speed, large volume of tests along with data transmission from PoN to authorities. Our data justify further development of the FNDP-NV-200nm OS and validation by authentic human specimens obtained from patients.