Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen

Abstract

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Chemical Modifications for a Next Generation of Nucleic Acid Aptamers

“…These aptamers target a variety of diseases, including myocardial ischemia, [113] metastatic renal cell carcinoma, [114] type 2 diabetes mellitus, [115] and thrombosis [116] . Apart from therapeutic usage, aptamers can also play an important role in disease diagnosis, for example, measuring the pulsatility of luteinising hormone, [117] analysing the level of various proteins in human serum [118] as well as detecting COVID‐19 antigens [119] . Nucleic acid aptamers are gaining more attention clinically, and will likely play increasingly important roles in future medicine.…”

Aptamers for SARS‐CoV‐2: Isolation, Characterization, and Diagnostic and Therapeutic Developments

“…Such assays possess advantages such as high sensitivity, ease of operation and high throughput [126] . There are now several reports of ALISAs for the detection of SARS‐CoV‐2 [43,50,113,127,128] . Svobodova et al .…”

“…Thiolated Apt1T was bound to a gold surface as the capture aptamer, and biotinylated Apt5B was used for detection. Upon the formation of the sandwich complex, poly‐HRP streptavidin was added to allow color generation based on a peroxide/TMB reaction [127] . The assay was able to detect down to 190 pM of S protein of SARS‐CoV‐2 in viral transport media, but only produced a sensitivity of 80 % and specificity of 40 % for clinical NPS specimens.…”

Sensitive and specific clinically diagnosis of SARS-CoV-2 employing a novel biosensor based on boron nitride quantum dots/flower-like gold nanostructures signal amplification

“…In the past two years, this severe acute respiratory syndrome leading to millions of deaths around the world and has become a global public health threat because of its rapid spread and significant mortality. It has also caused serious economic and social consequences and losses ( Pan et al, 2021 ; Svobodova et al, 2021 ; Verma et al, 2020 ). The efficient and smart diagnostic tests can have a significant impact on the management of the Covid-19 epidemic, enable rapid detection and isolation even before symptoms manifest ( Khan et al, 2020 ; Qiu et al, 2020 ; Udugama et al, 2020 ).…”