(Invited) A Biosensor Platform for on-Site Virus Visualization

Abstract

Introduction The detection of small amounts of specific targets such as toxins, pathogens, and viruses has become increasingly important since the SARS-CoV-2 pandemic. Particularly, the on-site virus detection by naked eye or camera, which allows direct and rapid visualization of the virus on a surface directly and rapidly, is one of the most appropriate ways to control and prevent from the infection. Currently available technologies for the virus detection such as quantitative PCR, ELISA, and lateral flow immunoassay are useful, but they require complicated sample extractions and sometimes bound/free (B/F) separation procedures, which makes the detection time-consuming and inconvenient. One realistic example of the on-site visualization is a forensic luminol blood test based on chemiluminescence because complicated detection procedures must be eliminated for the on-site visualization. The forensic test is based on the peroxidase activity of hemoglobin for the chemiluminescence emission upon the spraying of hydrogen peroxide solution. Therefore, we focused on generation of hydrogen peroxide in the proximal of virus and the associated emission of chemiluminescence, which enables us to the on-site visualization.To realize the localized hydrogen peroxide generation and successive chemiluminescence emission, we designed sequential enzymatic reactions using oxidase and peroxidase to be occurred only on the viral surface. In detail, we prepared antibody-enzyme complex (AEC) composed of oxidase and anti-virus antibody as a donor, and peroxidase-based molecules with virus-recognition ability as an acceptor. In the presence of virus, the donor and the acceptor bind to the virus, and a hydrogen peroxide relay from the donor and the acceptor on the viral surface will lead to the detection and visualization only after mixing of reagents (Fig.). Here, glucose oxidase (GOx) was used as a model donor enzyme because the substrate, glucose, is safe and highly stable, and conveniently prepared the AEC by SpyCatcher/SpyTag system, a protein coupling module for biosensing1). As the acceptor, bispecific aptamer targeting virus and peroxidase-based molecules was used. Then, we validated the utility of the platform by applying it to the detection and visualization of inactivated SARS-CoV-2. Methods The recombinantly produced SpyTag (ST)-fused anti-SARS-CoV-2 surface protein single-chain variable fragment (scFv-ST) and SpyCatcher (SC)-fused GOx (SC-GOx) were equivalently mixed and incubated at 4℃ to prepare the homogenous AEC. The bivalent AEC can be prepared because the GOx forms homo-dimer. The enzymatic activity and affinity were evaluated. Then, we constructed the detection systems using different peroxidase-based molecules as acceptors, and applied them to inactivated SARS-CoV-2 detection and visualization using a conventional smartphone camera. Results & Discussion SDS-PAGE analysis revealed that the AEC was prepared by only mixing of scFv-ST and SC-GOx via the SpyCatcher/SpyTag reaction. The AEC showed high binding ability, and K D values of scFv-ST and the AEC were 32 nM and 19 nM, respectively. This affinity enhancement was caused by the bivalency of the AEC. The AEC showed 72% of oxidase activity compared to the original GOx, suggested that the AEC retained sufficient activity for the signal generation as the donor.As the acceptor, myoglobin (Mb) and a bispecific aptamer composed of Mb aptamer2) and SARS-CoV-2 surface protein binding aptamer were used. The peroxidase activity of Mb is relatively weak, so a reduced decreased background signal is expected. The bispecific aptamer showed binding abilities to Mb and inactivated SARS-CoV-2.Using these detection components, we successfully detected inactivated SARS-CoV-2 by only mixing of the components, and the linear detection range met the clinically required level. Owing to the sequential enzymatic reactions only on the viral surface, the rapid detection system without any washing procedures was established. By exchanging a set of antibody and aptamer, it was applied to inactivated influenza A virus detection.Finally, hemin and hemin-binding aptamer were used as the acceptor and applied it to the inactivated SARS-CoV-2 visualization. As a result, not only the viruses in a 96-well plate but also on a nitrocellulose membrane was visualized by luminol-derived chemiluminescence through a conventional smartphone camera. This was a first report of the on-site virus visualization. In addition, the use of sequential enzymatic reaction provides the signal amplification when applied to electrochemical detection3), leading to more rapid and convenient detection using established portable devices. This system can be a platform for control of infection in public space in the future. References 1) D. Miura et al., Talanta, 234, 122638 (2021).2) K. Tsukakoshi et al., Nucleic Acid Res., 49, 6069-6081 (2021).3) B. Jeong et al., Anal. Chem., 85, 1784-1791 (2013). Figure 1