Citation

  • Authors: Pinals RL. et al.
  • Year: 2021
  • Journal: Nano Lett 21 2272-2280
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: HEK-293
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293, 293
    2. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T

Method

Synthesis and purification of SARS-CoV-2 VLPs To prepare the SARS-CoV-2 VLPs, two plasmids pcDNA3.1-Spike and pIRES2-MNE were synthesized based on the sequence of the Wuhan-Hu-1 strain. The spike protein was stabilized with the furin cleavage (residues 682-685) abrogated and the consecutive residue 986 and 987 substituted with prolines. The VLPs were synthesized by co-transfecting HEK293 or HEK293T cells with plasmids. To generate VLPs without S protein, cells were transfected with pIRES2-MNE only. The harvested supernatant was first concentrated with a 100 kDa MWCO centrifugal filter (Pall Corporation) then laid over discontinuous 20%-60% sucrose or Opti-prep (BioVision Inc.) gradients followed with centrifugation at 30,000 rpm for 4 hours. Purified VLPs were resuspended in PBS pH 7.4 and frozen at -80°C for storage.

Abstract

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.

Pubmed