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bioRxiv · 10.1101/2021.05.09.443341

Rapid Electronic Diagnostics of Ebola Virus with Synthetic Nanobody-Conjugated Gold Nanoparticles

Chen, X.·Kang, S.·Ikbal, M. A.·Zhao, Z.·Zuo, J.·Yao, Y.·Gu, L.·Wang, C.
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Abstract

Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays to be widely implemented at a high frequency. Here we present a generalizable assay platform, nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED), demonstrated in the detection of Ebola and COVID-19 antigens. To efficiently generate high-quality affinity reagents, synthetic nanobody co-binders and mono-binders with high affinity, specificity, and stability were selected by phage display screening of a vastly diverse, rationally randomized combinatorial library, bacterially expressed and site-specifically conjugated to gold nanoparticles (AuNPs) as multivalent in-solution sensors. Without requiring fluorescent labelling, washing, or enzymatic amplification, these AuNPs reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or simple electronic circuitry. With nanobodies against an Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (limit of detection of [~]10 pg/mL for sGP and [~]40 pg/mL for RBD in diluted human serum), a high specificity, and a large dynamic range ([~]7 logs). Unlike conventional assays where slow mass transport for surface binding limits the assay time, Nano2RED features fast antigen diffusion at micrometer scale, and can be accelerated to deliver results within a few minutes. The rapid detection, low material cost (estimated < $0.01 per test), inexpensive and portable readout system (< $5 and < 100 cm3), and digital data output, make Nano2RED particularly suitable for screening of patient samples with simplified operation and accelerated data transmission. Our method is widely applicable for prototyping diagnostic assays for other antigens from new emerging viruses.

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Chen, X., Kang, S., Ikbal, M. A., Zhao, Z., Zuo, J., Yao, Y., Gu, L., Wang, C.. 2021-05-10. Rapid Electronic Diagnostics of Ebola Virus with Synthetic Nanobody-Conjugated Gold Nanoparticles. https://doi.org/10.1101/2021.05.09.443341

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