Extraction of Viral Nucleic Acids with Carbon Nanotubes Increases SARS-CoV-2 Quantitative Reverse Transcription Polymerase Chain Reaction Detection Sensitivity

Abstract

The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using “capture” ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.

Carbon nanotubes in COVID-19: A critical review and prospects

“…These CNTs also facilitate the extraction of nucleic acids directly from the human saliva with nearly comparable efficacy as the commercially available DNA/RNA extraction kits, obviating the necessity for additional purification of the biofluids. The use of CNTs in the extraction of viral nucleic acids allows for high-yield and high-sensitivity identification of the SARS-CoV-2 nucleic acids, relying less on chemicals that are subject to change by constraints in the supply chains [ 50 ].…”

Multifunctional carbon nanomaterials for diagnostic applications in infectious diseases and tumors

“…ssDNA can adsorb to the surface of CNs by various interactions of the exposed bases with CNs (including π-π stacking, hydrophobic interactions, hydrogen bonding, and van der Waals forces), while the nucleobases of dsDNA are shielded by phosphate structures with a negative charge and have a low affinity for CNs [ 158 ]. Jeong and coworkers developed a method for yield extraction of SARS-CoV-2 nucleic acids using captured ssDNA sequences attached to the surface of SWCNTs, for direct downstream detection by qRT-PCR [ 159 ]. With the complementary binding of captured ssDNA to the target and reversible aggregation of SWCNT induced under acidic conditions, viral RNA can be extracted in high yield by subsequent adjustment of solution pH.…”

Nanotechnology-based approaches in the fight against SARS-CoV-2

“…Wang's group [48] have recently exploited modified selenium nanostructures to prepare a lateral flow immunoassay kit that can detect anti-SARS-CoV-2 antibodies (IgG and IgM) in human serum, with naked-eye in only 10 minutes. The researchers tested the bioassay in 90 COVID-19-diagnosed patients and 263 non-infected controls and reported 93.33% sensitivity and 97.34% specificity.…”

“…In the place of commonly used gold nanostructures in biosensor to detect SARS-CoV-2, some research groups used carbon nanotubes [41] , [48] , graphene [49] , [50] , and graphene-based (graphene oxide or reduced graphene oxide) [51] nanomaterials due to carbon nanotube's and graphene's high conductivity and biocompatibility [52] , [53] , and benefit of lower cost [29] . Further, the modification of graphene's surface with functional groups imparts the biosensor with very high sensitivity and ability to recognize biological molecule [54] .…”