Novel luminescent biosensor detects SARS-CoV-2 spike protein using cellphone camera
In a recent study published on bioRxiv* pre-print server, researchers developed a novel Bto find Aactivated Jandem fractionated enzyme (BAT) biosensor for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S).
credit: NIAID” class=”rounded-img” src=”https://d2jx2rerrg6sh3.cloudfront.net/images/news/ImageForNews_717032_16554371467354072.jpg” srcset=”https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/2000/src/images/news/ImageForNews_717032_16554371467354072.jpg 2000w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/1950/src/images/news/ImageForNews_717032_16554371467354072.jpg 1950w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/1750/src/images/news/ImageForNews_717032_16554371467354072.jpg 1750w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/1550/src/images/news/ImageForNews_717032_16554371467354072.jpg 1550w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/1350/src/images/news/ImageForNews_717032_16554371467354072.jpg 1350w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/1150/src/images/news/ImageForNews_717032_16554371467354072.jpg 1150w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/950/src/images/news/ImageForNews_717032_16554371467354072.jpg 950w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/750/src/images/news/ImageForNews_717032_16554371467354072.jpg 750w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/550/src/images/news/ImageForNews_717032_16554371467354072.jpg 550w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113911/ri/450/src/images/news/ImageForNews_717032_16554371467354072.jpg 450w” sizes=”(min-width: 1200px) 673px, (min-width: 1090px) 667px, (min-width: 992px) calc(66.6vw – 60px), (min-width: 480px) calc(100vw – 40px), calc(100vw – 30px)” style=”width: 2000px; height: 1491px;” width=”2000″ height=”1491″/>Study: A single-component luminescent biosensor for SARS-CoV-2 spike protein. Image credit: NIAID
All discovery and translational biomedical research primarily depends on antibody-based technologies for the detection of small molecule and protein analytes. These technologies have three limitations, as follows:
i) antibodies need mammalian expression systems for production,
ii) most antibody-based assays require chemical modification to immobilize the antibodies, and
iii) they require a minimum of two unique antibodies to function.
Single-component luminescent biosensors based on the split luciferase enzyme are simpler to produce, highly sensitive, and easier to use. Moreover, they only need one camera for detection and are suitable for multiple analysis formats. Compared to other single-component rigs, they do not rely on large conformational changes in the binding module or competing with a tethered lure.
About the study
In the current study, researchers deployed a single-component, luminescent BAT biosensor to detect the SARS-CoV-2 S protein in several assay formats. BATs consist of an antigen-binding module and a split enzyme, NanoBiT, which fuse in tandem to the N and C termini of the binding module.
The researchers selected the computer-designed long-chain basic subunit 1 (LCB1) of serine palmitoyltransferase as the binding module. LCB1 binds to the receptor binding domain (RBD) of SARS-CoV-2 protein S with a dissociation constant (KD) from 10-10. Additionally, LCB1 is rigid, thermally stable, and has no disulfide bonds to confuse purification. Its N and C ends are approximately 25 angstroms (Å) apart and in opposite directions, creating a rigid scaffold to minimize the effective concentration of the fused luciferase moieties.
Signal-to-noise ratio (S/N) multiplied by signal change (SN) indicated the performance for each linker composition. The researchers plotted the performance obtained based on the detection of a nano-molar (nM) recombinant S in phosphate-buffered saline (PBS). The molecule representing the best combination of linkers was named S-BAT.
The team also generated a point mutant in the S-BAT binding module to Asparagine30 to form S-BAT*. This molecule removed salt bridges with Lysine417 and Arginine403 in the S RBD, exhibited low background activity but showed minimal activation upon incubation with recombinant S or RBD, even at saturating concentrations .
Design of a Binding Activated Tandem Fractionated Enzyme (BAT) Biosensor for the SARS-CoV-2 Spike Protein. a) BATs consist of a binding module (B, gray) for antigen (red) and a split enzyme (1 & 2, Blue). The split enzyme is fused in tandem to the N and C termini of the binding module. A low effective concentration of 1 and 2 in the “OFF” state limits background activity. The steric conflicts induced by the binding between the antigen and the attached fractionated enzyme components increase the effective concentration by 1 and 2, resulting in reconstitution to the “ON” state. b) A model of an activated BAT (left) composed of SmBiT (light blue, cartoon), the mini protein LCB1 (grey, cartoon) and LgBiT (blue, cartoon) bound to the binding domain SARS-CoV-2 spike protein receptor (RBD, red, surface). The lengths of Linker1 and Linker 2 (left, black, dotted) are likely to control the effective concentration of SmBiT and LgBiT in the absence of the binder. c) A heat map of BAT performance as a function of linker length. Performance (signal to noise (S/N) multiplied by magnitude of signal change (SN)) for each linker composition is plotted against detection of 1 nM recombinant Spike in phosphate buffered saline (PBS) ). The best combination of linkers was a single amino acid (Gly) for linker 1 and a linker of length 0 for linker 2. This molecule is named S-BAT. d) Asp 30 in LCB1 (gray, cartoon) makes critical contacts with Arg 403 and Lys 417 in the RBD of the SARS-CoV-2 spike protein (red, cartoon). Mutation of Asp 30 to Ala results in S-BAT* which is not activated by recombinant Spike (middle panel) or RBD (right panel). Luminescence is plotted in arbitrary units (AU) against antigen concentration (molar, M) for individual replicate samples (n=3) in a representative experiment.
The S-BAT sensor was functional in several test formats. When adsorbed on paper, S-BAT showed a change in luminescence intensity as a signal. This adsorption-based immobilization approach required no chemical modification of the protein reagent. The team also demonstrated the performance of BATs in the Lateral Flow Analysis (LFA) format. In this S-BAT format, Cysteine16-PEG11-Biotin again detected 1 nM of recombinant S.
An enzyme immunoassay (ELISA) requires several antibody reagents and involves several development steps. In this format, S-BAT and Cysteine16-PEG11-Biotin detected low nM concentrations of antigens by imaging within 10 minutes. The authors immobilized biotinylated S-BAT constructs on streptavidin agarose beads against 10 nM recombinant S. The results were analyzed using a charge-coupled device (CCD) camera which showed that PEG11 linkers gave the highest signal and conjugation to Cysteine16 gave the highest S/N ratio.
phone. a) Computer-aided design (CAD) model of a mobile phone case (left) and cover (right) for low-light chemiluminescence imaging.” class=”rounded-img” src=”https://d2jx2rerrg6sh3.cloudfront.net/images/news/ImageForNews_717032_16554370792467817.jpg” srcset=”https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/1258/src/images/news/ImageForNews_717032_16554370792467817.jpg 1258w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/1250/src/images/news/ImageForNews_717032_16554370792467817.jpg 1250w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/1050/src/images/news/ImageForNews_717032_16554370792467817.jpg 1050w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/850/src/images/news/ImageForNews_717032_16554370792467817.jpg 850w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/650/src/images/news/ImageForNews_717032_16554370792467817.jpg 650w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20220616113800/ri/450/src/images/news/ImageForNews_717032_16554370792467817.jpg 450w” sizes=”(min-width: 1200px) 673px, (min-width: 1090px) 667px, (min-width: 992px) calc(66.6vw – 60px), (min-width: 480px) calc(100vw – 40px), calc(100vw – 30px)” style=”width: 1258px; height: 406px;” width=”1258″ height=”406″/>
Detection of SARS-CoV-2 protein S using S-BAT and mobile phone. a) Computer-aided design (CAD) model of a mobile phone case (left) and cover (right) for low-light chemiluminescence imaging.
Moreover, S-BAT did not require purification. The team used E.coli without affinity purification tag to express S-BAT and S-BAT*. A chemical lysis step followed by centrifugation and filtration yielded the crude lysate which could detect recombinant S with sensitivity and specificity at par with purified protein reagents.
S-BAT also worked robustly as a component of complex mixtures, such as when introduced into yeast growth medium S.cerevisiae. The filtered yeast culture medium, S.cerevisiae gave sufficient signal to detect nM recombinant S after 24 hours of induction of S-BAT expression. Even in HEK-293T cells expressing full-length SARS-CoV-2, S-BAT or S-BAT* could detect and produce a signal based on S expression and LCB1 binding integrity.
Notably, the BATs platform also reliably detected the live and cultured SARS-CoV-2 WA1 strain and the Delta variant (B1.617.2). However, S-BAT could not detect the mutated SARS-CoV-2 Omicron variant at K417 and N501 in protein S compared to the ancestral strain WA1.
Overall, S-BAT has emerged as a promising diagnostic reagent for the assay of pseudoviruses, cultured viruses, and protein subunits of coronavirus disease 2019 (COVID-19) vaccines based on the detection of SARS-CoV-2 S antigen. It also showed high versatility and sensitivity in several assay formats and was synthesizable at low cost.
In the current work, the authors used an expensive and unstable reporter enzyme system, Furimazine. They have focused on adapting BAT to incorporate different reporter enzyme systems, such as peroxidases, alkaline phosphatase, or other non-proprietary luciferase enzymes. Such improvisations, they say, could help adapt the existing BAT platform to more robust substrate classes and other antigens and give them the ability to produce luminescent and colorimetric readouts.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.
- A single-component luminescent biosensor for the SARS-CoV-2 spike protein, Matthew Ravalin, Heegwang Roh, Rahul Suryawanshi, G. Renuka Kumar, John E. Pak, Melanie Ott, Alice Y Ting, bioRxiv pre-print 2022, DOI: https://doi.org/10.1101/2022.06.15.496006, https://www.biorxiv.org/content/10.1101/2022.06.15.496006v1