Development of Nanobody-Displayed Whole-Cell Biosensors for the Colorimetric Detection of SARS-CoV-2

Abstract

The accurate and effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to preventing the spread of infectious diseases and ensuring human health. Herein, a nanobody-displayed whole-cell biosensor was developed for colorimetric detection of SARS-CoV-2 spike proteins. Serving as bioreceptors, yeast surfaces were genetically engineered to display SARS-CoV-2 binding of llama-derived single-domain antibodies (nanobodies) with high capture efficiency, facilitating the concentration and purification of SARS-CoV-2. Gold nanoparticles (AuNPs) employed as signal transductions were functionalized with horseradish peroxidase (HRP) and anti-SARS monoclonal antibodies to enhance the detection sensitivity. In the presence of SARS-CoV-2 spike proteins, the sandwiched binding will be formed by linking engineered yeast, SARS-CoV-2 spike proteins, and reporter AuNPs. The colorimetric signal was generated by the enzymatic reaction of HRP and its corresponding colorimetric substrate/chromogen system. At the optimal conditions, the developed whole-cell biosensor enables the sensitive detection of SARS-CoV-2 spike proteins in a linear range from 0.01 to 1 μg/mL with a limit of detection (LOD) of 0.037 μg/mL (about 4 × 108 virion particles/mL). Furthermore, the whole-cell biosensor was demonstrated to detect the spike protein of different SARS-CoV-2 variants in human serum, providing new possibilities for the detection of future SARS-CoV-2 variants.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications

“…In the presence of SARS-CoV-2 S proteins, the sandwiched binding was formed by interacting engineered yeast, SARS-CoV-2 S proteins, and reporter AuNPs. The colorimetric signal was obtained by the catalytic reaction of HRP, resulting in the developed whole-cell biosensor that enables the sensitive detection of SARS-CoV-2 S proteins with an LOD of 0.037 μg/mL, approximately 4 × 10 8 virion particles/mL [ 177 ].…”

“…AuNP aggregation and sedimentation assay mechanism was characterized with colorimetric readout and structural analysis for quantitative assessment of antigen concentration. Reprinted with permission from [ 177 , 181 ]. Copyright © 2023 American Chemical Society and Copyright © 2022 Elsevier.…”

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System

“…This trans -cleavage mechanism has been leveraged for the creation of many relevant CRISPR-Cas12a-based biosensors, , including for the detection of parasitic infections, bacterial infections, drug-resistant bacteria, and more. These systems often utilize fluorescence as a reliable signal transduction method. , While CRISPR-based fluorescent biosensors have successfully been developed for the detection of bacteria, complicated probe designs and expensive fluorescent quenchers have hindered the true portability and affordability for POC detection. − Additionally, standard fluorescent quencher (FQ) probes can cause steric hindrance, which can lower the trans -cleavage activity thus lowering sensitivity . Recently, nanomaterials have demonstrated the ability to offer simpler and more accessible options for fluorescent quenching …”

“… 25 , 26 27 While CRISPR-based fluorescent biosensors have successfully been developed for the detection of bacteria, complicated probe designs and expensive fluorescent quenchers have hindered the true portability and affordability for POC detection. 28 − 30 Additionally, standard fluorescent quencher (FQ) probes can cause steric hindrance, which can lower the trans -cleavage activity thus lowering sensitivity. 31 Recently, nanomaterials have demonstrated the ability to offer simpler and more accessible options for fluorescent quenching.…”

Enzyme-free electrochemical sensor platforms based on transition metal nanostructures for clinical diagnostics

“…1–3 The high-performance detection of emergent biomarkers is of crucial importance in several arenas, encompassing the healthcare industry, the clinical medicine and diagnostics sectors, and food safety. 4–6 Numerous biosensor platforms were established for the identification and measurement of biomarkers, such as lactic acid (LA), glucose (GLU), NADH, hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO), utilizing either in vitro or in vivo studies. 7–10 In particular, potent lactic acid and glucose sensing platforms are especially in great demand in a variety of industries, including those involved in clinical analysis, biomedicine, biological, food, cosmetics, pharmaceuticals, leather, sports, and chemical industries.…”