ABSTRACT
Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays for wide implementation at a high frequency. This paper presents a design for an in-solution assay pipeline, featuring nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED) of Ebola and COVID-19 antigens. Synthetic nanobody binders with high affinity, specificity, and stability are selected from a combinatorial library and site-specifically conjugated to gold nanoparticles (AuNPs). Without requiring any fluorescent labelling, washing, or enzymatic amplification, these multivalent AuNP sensors reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or portable electronic circuitry. With Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (the limit of detection of â¼10 pg /mL, or 0.13 pM for sGP and â¼40 pg/mL, or â¼1.3 pM for RBD in diluted human serum), a high specificity, a large dynamic range (â¼7 logs),and fast readout within minutes. The rapid detection, low material cost (estimated <$0.01 per test), inexpensive and portable readout system (estimated <$5), and digital data output, make Nano2RED a particularly accessible assay in screening of patient samples towards successful control of infectious diseases.