Title：Synthetic nanobody-functionalized nanoparticles for accelerated development of rapid, accessible detection of viral antigens

Journal：《Biosensors and Bioelectronics》

Link：https://doi.org/10.1016/j.bios.2022.113971

Reporter：Wei Juan, master of 2020

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 ~10 pg /mL, or 0.13 pM for sGP and ~40 pg/mL, or ~1.3 pM for RBD in diluted human serum), a high specificity, a large dynamic range (~7 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.

In recent years, viral infectious diseases have emerged many times. In order to cope with the possible emergence of highly infectious and highly lethal viral infectious diseases in the future, it is necessary to accelerate the design, development and verification of the diagnostic process.

Current diagnostic methods rely on the detection of genetic (or molecular) antigens or serological (antibody) markers. Genetic diagnosis uses DNA sequencing polymerase amplification assays or more recently CRISPR technology. For example, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays are considered the gold standard due to their high sensitivity In contrast, antigen and antibody testing allows for more rapid, affordable and accessible testing without the need for complex sample preparation or amplification. Therefore, the detection method suitable for the monitoring and timely isolation highly infectious individuals, particularly in the clinical environment. Although antibodies (IgM) detection has been used in disease diagnosis, but its low predictability, more suitable for in the study of immune response. In contrast, viral protein antigen detection for diagnosis of patients with symptoms provide reliable field test solutions. In addition, antigen testing, due to its rapid ease of operation and low cost, can be deployed in large numbers at high frequency for real-time surveillance, which is considered a crucial factor in disrupting viral transmission chains.

Enzyme-linked immunosorbent assay (ELISA) and side-flow immunoassay (LFIs) are the main tools for antigen and antibody analysis. However, ELISA is complicated with many washing steps, several hours of incubation period before reading, and a readout system that depends on substrate conversion and luminescence recording. Deployment in the high-throughput screening high-throughput ELISA need automated liquid handling system to coordinate the work of complex process, it is not ideal for portable applications. LFIs be more easily in the laboratory using external environment, but compared with ELISA, its sensitivity is low, usually so poor accuracy. To establish a fast easy to operate, it is necessary to detect virus antigens with low cost and high frequency and mass deployment in the field.

1.     Nanobody co-binder selection for AuNP functionalization

Fig. 1. Overview of Nano2RED assay development and characterizations.

Fig. 2. Identification and characterization of antigen-specific nanobody binders

Authors generated nanobody co-binders against target antigens (Fig. 1a) using a fast, robust protocol, including the phage display selection of the combinatorial nanobody library, parallel bacterial protein production, co-binder validation, and AuNP functionalization. They assessed clonal diversity and co-binding abilities of candidates enriched in different biopanning rounds (Fig. 2a). The two top co-binder pairs, termed sGP7–sGP49 and RBD8–RBD10, were bacterially expressed and purified (Fig. 2b). Their equilibrium dissociation constants (K D ) were measured to be in the nanomolar range by Bio-Layer Interferometry (BLI) (Fig. 2c) and the co-binding activities were validated by ELISA (Fig. 2d) and BLI (Fig. 2e). Lastly, nanobodies were biotinylated with E. coli biotin ligase (BirA) as previously reported and then loaded to streptavidin-coated AuNPs.

In this assay design, AuNPs densely coated with biotinylated nanobodies allow multivalent antigen sensing (Fig. 1a and b) known to significantly enhance antigen binding compared to the monovalent binding. Further, the multivalent binding also facilitates AuNP aggregation and subsequent precipitation, producing antigen-concentration-dependent signals within minutes. The sensing principle is that AuNPs are initially uniformly dispersed in colloids in the absence of non-specific particle to particle interactions, showing a red color of local surface plasmon resonance (LSPR) extinction After mixing with viral antigens, multiple AuNPs are bound together by antigen-nano antibodies and gradually form aggregates, which gradually expand LSPR extinction and pellet formation when gravity exceeds fluid resistance (Figure 1c-d). This results in increased transparency of the AuNP colloid, which can then be further quantified with a spectrophotometer or simple electronic device on the well plate.

2.     Colorimetric and spectrometric sensing of sGP

The authors first optimized the diameter and finally selected 80 nm. Firstly, AuNP functionalized with nanobody sGP49 was used to detect sGPS. The results were shown in FIG. 3, it could detect sGP from 10 pM to 100 nM, which supports clinically relevant Ebola detection from patients’ blood (sub-nM to μM). They also found the 10 nM sGP could be easily distinguished from NC sample at a broad temperature range from 20 to 70 ℃ (Fig. 3h). This indicates this assay is stable at ambient temperatures without serious concerns of performance degradation during transportation or storage, which is very important for mass screening.

Fig. 3. Ebola sGP sensing using mono-binder antibody sGP49 by incubation.

3.     Rapid sGP detection with a portable, electronic readout device (Nano2RED)

3 h incubation was effective for AuNP ligation and precipitation, and was shorter than ELISA and much better than many RT-PCR assays. However, rapid diagnosis, which is less than 30 minutes, was more advantage. Therefore, the authors further investigated rate-limiting sensing mechanisms to reduce detection time using sGP(10 nM) as antigen and found that after vortex mixing, the color contrast was high enough to be immediately discernible to the naked eye, requiring minimal incubation (Fig. 4 h) and all procedures including sample collection pipetting centrifugation vortex mixing readings, and this rapid test protocol could be completed in a few minutes.

Fig. 4. Rapid and electronic detection of sGP using mono-binder antibody sGP49 with improved sensing performance.

4.     Detection of sGP and RBD in serum and blood

The authors further evaluated the use of co-bound nanobodies in sGP and RBD sensing (Figure 5), namely sGP49/sGP7 for sGP and RBD8/ RBD10 for RBD (Figure 2), and performed rapid detection in PBS FBS human blood pool (HPS) and whole blood (WB).

In addition, the authors reveald the importance of systematic analytical design strategies, from molecular binding to signal transduction and readout, to optimize antigen detection, which is evident with cobinder pairs increasing LOD by 10-100-fold compared to single binders (sGP49, K D 4.6 nM).

Fig. 5. Co-binders for rapid sGP and RBD detection in different buffers.

In summary, this paper demonstrated a generalizable and rapid assay design and pipeline that shows high sensitivity (limit of detection for sGP: 10 pg /mL, 0.13 pM; RBD detection limit is 40 pg/mL, 1.3 pM), high specificity, large dynamic range (~7 log), and rapid readout within minutes. This method is very feasible for accurate antigen quantification and early infection detection. It can also be used for high-frequency diagnosis in homes or clinics, as well as areas with limited resources, which can greatly enhance control of disease transmission. In the future, digital data formats will reduce human intervention in data compilation and reporting while facilitating fast and accessible data analysis. Nano2RED may find immediate use in antigen and antibody testing during the current COVID-19 pandemic, as well as in preparation for unforeseen new outbreaks in the future.