Amplification-Free SARS-CoV-2 Detection Using Nanoyeast-scFv and Ultrasensitive Plasmonic Nanobox-Integrated Nanomixing Microassay

Abstract

The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/μL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.

Silver nanotriangle array based LSPR sensor for rapid coronavirus detection

“…In addition, the viral particles used in Refs. [14] , [49] are SARS-CoV-2 pseudotyped viral particles or inactivated virus, not the real virus. Our estimation on the number of viral particle binding shows that at the LOD, every AgNT has an average of one viral particle bonded, which further confirms the reliability of our detection.…”

Surface-enhanced Raman scattering-based immunoassay for severe acute respiratory syndrome coronavirus 2

“…SERS is an effective technique for the sensitive detection of biomolecules, and various SERS-based immunoassays have thus been developed for the detection of infectious viruses ( Saviñon-Flores et al, 2021 ). Following the emergence of the COVID-19 pandemic, several SERS-based methods for SARS-CoV-2 have been applied ( Zhang et al, 2021 ; Wu et al, 2022 ; Yang et al, 2021 ; Yu et al, 2021 ; Srivastav et al, 2021 ; Sanchez et al, 2021 ; Payne et al, 2021 ; Liu et al, 2021 ; Li et al, 2021 ; Huang et al, 2021 ; Gao et al, 2021 ; Chen et al, 2021 ; Chen et al, 2021 , 2021 ; Huang et al, 2021 , 2021 ; Pramanik et al, 2021 ; Zavyalova et al, 2021 ). However, the current magnetically assisted SERS immunoassay is the only assay developed so far that facilitates the on-site diagnosis of the samples of COVID-19 patients.…”

“…However, recent advancements in SERS have led to the development of sophisticated sensing methods that facilitate precise analysis and generation of high-quality SERS-active nanostructures ( Hwang et al, 2019 ; Shin et al, 2018 ). In particular for the combat with COVID-19, SERS technology has not only exhibited less time-cost advantages than PCR for detection SARS-CoV-2 and antibodies, but also exhibited more advantages such as identifying infectiousness of SARS-CoV-2 ( Peng et al, 2021a , Peng et al, 2021b ; Zhang et al, 2021 ; Wu et al, 2022 ; Yang et al, 2021 ; Yu et al, 2021 ; Srivastav et al, 2021 ; Sanchez et al, 2021 ; Payne et al, 2021 ; Liu et al, 2021 ; Li et al, 2021 ; Huang et al, 2021 ; Gao et al, 2021 ; Chen et al, 2021 ; Chen et al, 2021 , 2021 ; Huang et al, 2021 , 2021 ; Pramanik et al, 2021 ; Zavyalova et al, 2021 ).…”

Next-Generation Molecular Discovery: From Bottom-Up In Vivo and In Vitro Approaches to In Silico Top-Down Approaches for Therapeutics Neogenesis

“…This phage library screening technique termed “PhageXpress” has been reported to require only a single biopanning step with AC-EHD flow, in combination with next-generation nanopore sequencing to achieve sufficient physical enrichment for isolation of target-binding phage clones. AC-EHD flow, used also in on-chip diagnostics [ 72 , 73 , 74 , 75 , 76 , 77 , 78 ], is proposed to reduce the “non-slip” property of fluid as it approaches a surface—a phenomenon described by Poiseuille’s law [ 79 ]—which can perturb microfluidic technologies. On-chip application of electrical potentials has also been used to produce digital microfluidics, where for example, electrowetting on dielectrics has been used to automate all steps—including counter selection—in multiple rounds of phage biopanning to produce a competent target-binding peptide [ 80 ].…”