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

  • Marketa Svobodova
    Marketa Svobodova
    INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
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  • , 
  • Vasso Skouridou
    Vasso Skouridou
    INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
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    Orcidhttps://orcid.org/0000-0002-9712-5429
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  • Miriam Jauset-Rubio
    Miriam Jauset-Rubio
    INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
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  • Irene Viéitez
    Irene Viéitez
    Rare Diseases & Pediatric Medicine Research Group, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-Uvigo, Vigo 36213, Spain
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    Orcidhttps://orcid.org/0000-0001-9241-0207
  • , 
  • Alberto Fernández-Villar
    Alberto Fernández-Villar
    Pneumology Service, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-Uvigo, Vigo 36213, Spain
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  • , 
  • Jorge Julio Cabrera Alvargonzalez
    Jorge Julio Cabrera Alvargonzalez
    Microbiology Service, Galicia Sur Health Research Institute (IIS Galicia Sur). SERGAS-UVigo, Vigo 36213, Spain
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  • Eva Poveda
    Eva Poveda
    Group of Virology and Pathogenesis, Galicia Sur Health Research Institute (IIS Galicia Sur)-Complexo Hospitalario Universitario de Vigo, SERGAS-UVigo, Vigo 36213, Spain
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  • , 
  • Clara Benavent Bofill
    Clara Benavent Bofill
    Laboratori Clinic ICS Camp de Tarragona, Hospital Universitari de Tarragona Joan XXIII, Avda. Dr. Mallafré Guasch, 4, Tarragona 43007, Spain
    More by Clara Benavent Bofill
  • , 
  • Teresa Sans
    Teresa Sans
    Laboratori Clinic ICS Camp de Tarragona, Hospital Universitari de Tarragona Joan XXIII, Avda. Dr. Mallafré Guasch, 4, Tarragona 43007, Spain
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  • Abdulaziz Bashammakh
    Abdulaziz Bashammakh
    Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 80215, Kingdom of Saudi Arabia
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  • Abdulrahman O. Alyoubi
    Abdulrahman O. Alyoubi
    Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 80215, Kingdom of Saudi Arabia
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  • , and 
  • Ciara K. O’Sullivan*
    Ciara K. O’Sullivan
    INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
    Institució Catalana de Recerca i Estudis Avancats (ICREA), Passeig Lluís Companys 23, Barcelona 08010, Spain
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-2603-2230
Cite this: ACS Omega 2021, 6, 51, 35657–35666
Publication Date (Web):December 15, 2021
https://doi.org/10.1021/acsomega.1c05521

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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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.

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Introduction

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Coronaviruses (CoV’s) are a group of highly diverse, enveloped, single-stranded, and positive-sense RNA viruses which cause diseases involving respiratory, enteric, hepatic, and neurological systems with a high range of severity among humans and animals. (1,2) To date, there are seven documented human CoV’s (HCoV’s). These include HCoV-NL63, HCoV-HKU1, HCoV-OC43, and HCoV-229E, which cause asymptomatic or mild respiratory infections. Severe acute respiratory syndrome (SARS)-CoV (SARS-CoV) and middle east respiratory syndrome (MERS)-CoV (MERS-CoV), on the other hand, are highly pathogenic and lethal HCoV’s. (3) The most recently recorded pathogenic HCoV is the novel SARS-CoV-2, previously named 2019-nCoV, which is the causative agent of the respiratory illness COVID-19, leading to millions of deaths around the world. (4,5)
SARS-CoV-2 contains four major structural proteins: the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. (6−8) S, E, and M are anchored on the viral surface envelope, whereas N is found inside the virion. Protein S is a typical class I fusion glycoprotein that undergoes proteolytic cleavage, resulting in two subunits S1 and S2. The receptor-binding domain (RBD) is part of the S1 subunit and it binds to a host receptor angiotensin-converting enzyme II (ACE2) on cells and then fuses the viral and host membranes through the S2 subunit. (9−11) The fundamental role of S protein in viral attachment, fusion, and entry to the cells renders it a key target for the diagnosis and treatment of SARS-CoV-2, (12) and it has indeed been the main protein target used in currently approved COVID-19 vaccines. (13)
The current COVID-19 diagnostic products can be grouped into serological and diagnostic tests. (14) Serological tests measure antibodies against SARS-CoV-2 in patients to test who may still be at risk and more broadly assess the prevalence of COVID-19 in a population. Diagnostic tests focus on nucleic acid or viral antigen detection. The standards for the diagnosis of COVID-19 are nucleic acid amplification tests (NAATs), including reverse transcription-polymerase chain reaction (RT-PCR), (15) transcription-mediated amplification (TMA), and loop-mediated isothermal amplification (LAMP). For the specific detection of SARS-CoV-2, diagnostic panels consisting of primers specific to SARS-CoV-2 are used and provide results in 2–3 h. Most of the available NAATs utilize primers selected from N, E, and S proteins and open reading frame 1 ab (ORF1ab) genes. (16,17) NAAT-based point-of-care (POC) tests are already available in the market offering easier handling and shorter turnaround times but still need to be used in physician offices or clinics. Moreover, molecular tests are expensive and cannot cover the massive testing needs for the control of the pandemic situation. Limited availability of NAAT testing capacity has hindered response effort even in well-funded health-care systems, while in low- and middle-income countries, the situation is even more complicated. Therefore, it is critical to develop alternative tests not limited to testing at large centralized facilities.
Antigen tests detect the presence or absence of the viral proteins (antigens) and offer an inexpensive solution for population-scale screening of SARS-CoV-2, particularly with lateral flow rapid tests. Currently, several rapid antigen tests are available in the market having received an emergency use authorization (EUA) from the United States Food and Drug Administration (FDA). (17−20) The majority of these tests are based on the detection of the N protein antigen. A recent study suggested the combined detection of the two antigens for more reliable results based on their finding that an S-based assay was more specific, whereas an N-based one was more sensitive for the detection of SARS-CoV-2 infections. (21) Due to the advantages they present compared to RT-PCR, rapid antigen tests could even substitute NAAT-based testing for screening purposes. Several studies demonstrated a high correlation between the antigen concentration and the viral load, especially in clinical samples with low cycle threshold (Ct) values corresponding to samples with a high viral load. (22−24) It has been reported that the concentration of the N protein antigen in symptomatic patient samples taken within 7 days of symptom onset correlates better with virus culture-based detection of the infectious virus than Ct values, (25) and thus, antigen tests can be used to screen for infectious individuals. (25) However, antigen tests with low sensitivity can produce false negative results for samples with a low viral load (high Ct values). (26,27)
Sandwich-type immunoassays have been established in the field of viral diagnostics for the detection and quantification of viral antigens, with their inherent characteristics of superior sensitivity and specificity, greatly reducing false-positive results. Taking lessons from the first SARS-CoV outbreak for which extremely sensitive sandwich ELISA assays targeting the nucleoprotein of the virus were developed, achieving detection limits of 50 pg/mL, (28) the first immunoassays developed for the detection of SARS-CoV-2 antigens were also based on the N protein. (29,30) In parallel, a variety of immunoassays and immunosensors, (31−37) ACE2 receptor-based biosensor, (37,38) or even glycan-based lateral flow assay (39) have also been reported in the literature for the detection of the S antigen, achieving limits of detection (LODs) from 1 fg/mL to 5 μg/mL.
Aptamers are considered as an alternative to antibodies and can be potentially used to improve currently available detection methods. Aptamers are synthetic nucleic acids selected via systematic evolution of ligands by exponential enrichment (SELEX), (40−42) forming three-dimensional structures allowing them to specifically bind to any kind of target molecules. They are inexpensive, stable, and versatile and can be easily modified and applied to virtually any kind of assay. The vast majority of aptamer selection strategies focus on obtaining at least one aptamer with high affinity and specificity, and there are a limited number of reports detailing aptamer-based sandwich assays. (43−45)
Recently, aptamers have been selected against the RBD of SARS-CoV-2 spike glycoprotein. (46) Two high binding affinity sequences were identified, aptamer 1 and 4, with affinity dissociation constant values (KD) of 3.1 and 13 nM, respectively. Truncated versions of these aptamers (aptamer 1C, 51 nt and aptamer 4C, 67 nt) were shown to maintain their high affinity with only slightly lower KD values of 5.8 and 19.9 nM for 1C and 4C, respectively. Aptamer 1C was later used for the development of a surface-enhanced Raman spectrometry (SERS)-based aptasensor for the detection of sub-picomolar concentrations of the spike protein, (47) while aptamer 4C immobilized on gold nanostars was exploited for spike protein detection using distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy, achieving an LOD of 1.7 fM. (48) Other high affinity spike protein-binding aptamers (KD in the low nanomolar range) have also been reported, (49−53) and in silico modeling has also been used to predict aptamers for binding the spike protein. (54−56)
Overall, these are the only studies reported to date regarding the development of S antigen-binding aptamers and the respective applications developed for the detection of SARS-CoV-2 infections.
The aim of this work was to develop an aptamer-based sandwich assay for the detection of the S antigen of SARS-CoV-2. We envisioned that once a microplate sandwich assay was established, its format could potentially be transferred to a POC-compatible format such as a lateral flow assay. To this end, all aptamer candidates previously reported, (46) six full-length and two shortened aptamer versions, were used in combinatorial experiments in order to identify pairs of aptamers able to detect the S1 antigen protein of SARS-CoV-2. Direct enzyme-linked aptamer assays (direct ELAAs) were initially performed to evaluate the affinity and specificity of the eight aptamers, followed by screening of different combinations of aptamers in the sandwich format to identify the best-performing aptamer pair in terms of sensitivity and specificity. Finally, a sandwich ELAA was developed using the best aptamer pair (Figure 1) and used for the detection of the spike antigen of SARS-CoV-2 in nasopharyngeal (NP) swab samples from COVID-19 patients.

Figure 1

Figure 1. Detection of the SARS-CoV-2 S antigen with the aptamer sandwich assay.

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Results and Discussion

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Affinity and Specificity of the Aptamers

With the COVID-19 pandemic caused by the SARS-CoV-2 virus still advancing more than a year since its outbreak and the global vaccination rates not reaching the desired level for achieving herd immunity, availability of SARS-CoV-2 diagnostic tools to implement the current monitoring and containment strategies is essential. Antigen tests have emerged as complementary tools to RT-PCR and typically detect the S or N viral proteins. The lower homology of S proteins among the known HCoV’s compared to the N protein renders the S protein as the most appropriate antigen for the development of sensitive and specific antigen tests. Viral antigen tests have recently emerged as key diagnostic tools that can implement the global efforts of monitoring and containment of the COVID-19 pandemic. (57) With the aim of developing an antigen test for SARS-CoV-2 based on the spike glycoprotein, previously reported spike protein RBD domain-binding aptamers (46) were exploited. The binding affinities of all full-length aptamers (1–6) and the two truncated versions (1C and 4C) for both S1 and RBD proteins of SARS-CoV-2 were initially evaluated using direct ELAAs. The KD values calculated for each aptamer and both proteins are shown in Table 1, while the binding curves can be found in Figure S1 (Supporting Information). All the KD values were observed to be in the low nanomolar range, demonstrating the high affinity of the aptamers. Slightly different KD values of aptamers 1 and 4 and their shortened versions 1C and 4C for the RBD compared to previously published results can be attributed to the different binding assays employed and the aptamer modifications. In the original report, (46) site-directed immobilization of the RBD protein on nickel beads was achieved via its his-tag, whereas random orientation is expected when the protein is adsorbed directly on a microtiter plate for the ELAAs. Moreover, the type of chemical modification added to the aptamer sequence could affect aptamer folding and therefore its binding properties. For all studied aptamers, higher affinities to S1 as compared to the RBD were observed. It is possible that the conformation of the S1 protein, which is larger compared to the RBD (76.5 kDa vs 26.5 kDa), is different after physical adsorption to the microtiter plate and could provide improved accessibility of the aptamer to the binding site on the RBD. Another factor that could contribute to these differences is the variability of the quality of the recombinant proteins and the organisms used for their expression. Similar results of higher affinity to S1 compared to the RBD were also reported for peptide binders selected recently against SARS-Cov-2 spike protein. (58)
Table 1. Affinity of the Aptamers for S1 and RBD SARS-CoV-2 S Protein Domains
 S1RBD
 KD (nM)R2KD (nM)R2
Apt110.2 ± 2.70.987539.0 ± 11.20.9903
Apt20.2 ± 0.10.97687.7 ± 1.40.9887
Apt35.3 ± 0.50.997610.9 ± 2.90.9706
Apt43.3 ± 0.70.986934.6 ± 17.80.9851
Apt511.3 ± 6.10.981029.8 ± 17.80.9713
Apt63.4 ± 1.10.987932.2 ± 7.30.9701
Apt1C8.1 ± 1.00.993921.0 ± 2.70.9916
Apt4C11.1 ± 2.70.962320.8 ± 5.40.9792
The specificity of an assay is a critical factor determining its analytical performance, particularly when screening for SARS-CoV-2 antigens. The potential cross-reactivity of the aptamers being evaluated in this work for the development of a SARS-CoV-2 antigen test with other HCoV’s was thus investigated, considering that the genome of SARS-CoV-2 shares 65–82.5% sequence homology with the other known HCoV’s such as HCoV-HKU1, HCoV-NL63, HCoV-229E, HCoV-OC43, SARS-CoV, and MERS-CoV. (59) As commented previously, the target protein of the SARS-CoV-2 antigen test is the spike glycoprotein of SARS-CoV-2. Taking into account that the protein in its prefusion state is assembled on the virion envelope by the S1 and S2 domains after proteolysis, as well as the limited commercial availability of recombinant RBD protein domains from the different HCoV strains, S1 was chosen as the most appropriate protein to be used from this point on. Hence, the binding of the aptamers to the S1 subunit of the other HCoV’s was evaluated by a direct ELAA (Figure 2). The lowest cross-reactivity to other HCoV’s was exhibited by aptamers Apt1 and Apt5. Apt1 appeared to be the most selective among the aptamers studied and it showed less than 20% relative binding to HCoV-NL63 and HCoV-229E. These are alpha CoV’s, and the RBD from SARS-CoV-2, which is a beta CoV, shares only 20% sequence homology. (60) Spike proteins from strains of the beta CoV genus, including MERS-CoV, HCoV-OC43, and HCov-HKU1, share higher sequence and structural similarities. (61) Apt1 showed cross-reactivity with S1 from these viruses (20–42% relative binding), suggesting that a similar binding site might be recognized. Surprisingly, no binding of Apt1 was detected to S1 from SARS-CoV which is the most recent common ancestor of SARS-CoV-2 (62) and with which it shares a 74% sequence identity within the RBD region. (63) The specificity of Apt5 followed a similar pattern with Apt1. The rest of the aptamers, however, showed higher cross-reactivity with the other CoV’s. Moreover, decreased specificity was observed for the truncated aptamers Apt1C and Apt4C as compared to their full-length counterparts despite maintaining their high affinity.

Figure 2

Figure 2. Specificity of the aptamers evaluated by direct ELAA. The relative binding of each aptamer was calculated using constant concentrations of aptamers (200 nM) and S1 proteins from the different HCoV’s (2 μg/mL). The error bars correspond to the standard deviation from duplicate measurements.

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Screening of Aptamer Pairs

The advantage of sandwich-type aptamer-based assays is that the combination of two aptamers can considerably improve the specificity and sensitivity of the assay. In order to find the most appropriate pair of aptamers for the development of the SARS-CoV-2 antigen test, pairwise experiments combining all eight aptamer candidates were carried out. Thiolated capture aptamers (1T–6T, 1CT, and 4CT) and biotinylated reporter aptamers (1B–6B) were thus combined in a sandwich format (Figure 1) using constant concentrations, and the results are shown in Figure 3. Nonspecific interactions between capture and reporter aptamers in the absence of protein were also evaluated. The majority of combinations tested resulted in the successful detection of S1 in a sandwich format, indicating that capture and reporter aptamers potentially bind to different binding sites on the target protein. Signals observed in the absence of S1 protein with the aptamer combinations of 4T with 2B, 5T with 3B, and 4CT with 5B could arise from partial sequence complementary, and consequently, these aptamer pairs were eliminated from the study.

Figure 3

Figure 3. Screening of aptamer pairs for the detection of SARS-CoV-2 S1 protein. Each thiolated capture aptamer (AptxT) was analyzed in combination with each of the biotinylated reporter aptamers (AptxB) using a constant concentration of S1. No protein was added for the control samples. The number refers to the number of the aptamer tested, T indicates the thiolated aptamer and B indicates the biotinylated aptamer.

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In search of the best-performing aptamer pair, the sensitivity of several combinations was then evaluated. Eight pairs were chosen based on both the specificity of the individual aptamers (Figure 2) and the relative binding they exhibited in the sandwich format (Figure 3). The pairs chosen were 1T/5B, 2T/1B, 3T/1B, 4T/1B, 5T/1B, 6T/6B, 1CT/6B, and 4CT/6B. S1 standard curves were thus constructed using the target protein in a range of 0.0032–50 μg/mL (equivalent to 0.042–654 nM), and the LODs were calculated as the bottoms of the curves fitted to the data using the four-parameter logistic model plus three times the standard deviation of the bottoms. As can be seen in Figure 4, high sensitivity was exhibited by all aptamer pairs tested, with LODs in the low nanomolar range (2.2–14.5 nM, equivalent to 0.17–1.11 μg/mL). EC50 values varied from 10.9–873.7 nM (0.83–66.84 μg/mL). The best-performing pair was 1T/5B with an LOD of 2.2 nM (170 ng/mL) and an EC50 of 10.9 nM (830 ng/mL). It has to be pointed out that the assays were not optimized at this point; therefore, further improvement of their performance is expected following optimization.

Figure 4

Figure 4. Sensitivity of the sandwich assays for SARS-CoV-2 S1 detection using different combinations of aptamer pairs.

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In addition to sensitivity, specificity is one of the most important parameters defining the performance of an analytical assay. For this reason, the response of the sandwich assays to S1 domains from all known HCoV’s was evaluated using the chosen aptamer pairs. As shown in Figure 5, the 1T/5B pair exhibited the highest specificity. The combination of Apt1 as a capture aptamer with Apt5 as a reporter aptamer decreased the cross-reactivity shown by the individual aptamers for HCoV-HKU1 and HCoV-OC43, confirming that sandwich formats can improve the specificity of analytical assays. However, some cross-reactivity with S1 from MERS-CoV was observed for the 1T/5B pair, similar to when the two aptamers were used individually. Cross-reactivity effects with MERS-CoV S-RBD protein were also observed with a near-infrared biosensor developed for SARS-CoV-2 S antigen detection using ACE2. (38) The same study also reported nonspecific signals from the SARS-CoV S protein and an influenza antigen. Since a cell membrane receptor was utilized as the biorecognition element, such interference from other viral antigens can be expected. In contrast, when specifically developed receptors such as aptamers and antibodies are used for bioassay design, cross-reactivity with other biomolecules can be prevented. Nevertheless, high antigen sequence and structural similarities can contribute to lower assay specificity even when monoclonal antibodies are used. (36) Consequently, the specificity exhibited by the 1T/5B pair was a decisive factor in choosing this pair for final assay development. The low cross-reactivity with the MERS-CoV antigen is not of critical importance since the occurrence of this virus in the population during the present pandemic is negligible.

Figure 5

Figure 5. Specificity of the sandwich assays using different aptamer pairs and S1 from different HCoV’s. Each protein was used at 2 μg/mL, and the error bars indicate the standard deviations from duplicate measurements.

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Sensitivity of the Apt1T/Apt5B Pair for Sandwich Detection of SARS-CoV-2 S1

As mentioned earlier, an initial screening of the aptamer pairs was performed in order to choose the best-performing pair in terms of sensitivity and specificity. To improve the performance of the sandwich assay based on the 1T/5B aptamer pair, the concentrations of both capture (Apt1T) and reporter (Apt5B) aptamers were optimized. A checkerboard assay was carried out first using 31.25–2000 nM Apt1T aptamer in combination with 7.8–250 nM Apt5B aptamer. The highest signals were obtained for the 1T capture aptamer at concentrations ≥250 nM in combination with the Apt5B reporter aptamer at concentrations ≥125 nM (Figure S2, Supporting Information). The sensitivity of the assays was thus evaluated using 125–500 nM Apt5B reporter aptamer, while the capture aptamer Apt1T was maintained at the highest concentration tested (2000 nM). The lowest LODs were calculated when 125–250 nM Apt5B was used, enabling the detection of as low as 270–280 pM (21–22 ng/mL) S1 (Figure S3, Supporting Information). This corresponds to the improvement of 1 order of magnitude compared to when nonoptimized assay conditions were employed.
Since the final objective of the study is detection of the S antigen in NP swab samples from individuals suspected of having been infected with SARS-CoV-2, the compatibility of the assay with the viral transport media typically used to collect these samples was evaluated. Serial dilutions of the target S1 protein were prepared in viral transport medium (VTM), and the assay was performed as described earlier with some modifications. The volume of the sample or the standard protein analyzed was 25 μL. As can be observed in Figure 6, the performance of the assay under these modified conditions in VTM was similar to the one conducted under optimized conditions. The LOD was 190 pM (15 ng/mL) compared to 270 pM (21 ng/mL) calculated for the assay in PBS-based buffer. This corresponds to the detection of 375 pg of S1 in VTM, more than an order of magnitude lower than the sensitivity achieved by a rapid antigen test based on a sandwich between ACE2 and a spike protein monoclonal antibody (<5 ng of S1). (37) Recent reports exploiting the aptamers selected by Idili et al., in electrochemical aptamer-based (EAB) sensors, report detectability over a concentration range of 760 pg/mL–76 ng/mL (64) and detection limits of as low as ag/mL, (65) but real saliva or NP samples are not tested. Cennamo et al. also used Song’s aptamers in a plasmonic D-shaped plastic optical fiber aptasensor, achieving a detection limit of 37 nM, but the sensor was not applied to the analysis of real samples. (66) Li et al. selected an aptamer against the wild-type and B.1.1.7 spike protein and demonstrated the use of the selected aptamers for the detection of a pseudotyped lentivirus in saliva samples, but again, no real samples from individuals with SARS-CoV-2 were analyzed. (51) In another work, the use of aptamer-functionalized gold nanoparticles was reported, achieving a detection limit of 10 nM, which is equivalent to 3540 genome copies/μL of inactivated SARS-CoV-2. (67) Using a 44-mer G-quadruplex-forming DNA aptamer they selected against the spike trimer antigen of SARS-CoV-2, Gupta et al. achieved a detection limit of 2 nM. (52) They used this aptamer for the analysis of 232 NP samples, achieving a highly discriminatory response between SARS-CoV-2-infected individuals from the noninfected ones with a sensitivity and specificity of 91 and 98%, respectively. (52)

Figure 6

Figure 6. Sensitivity of the sandwich assay based on the Apt1T/Apt5B aptamer pair in aptamer binding buffer and in VTM.

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The sensitivity of the assay compares well with other studies reported in the literature, exploiting antibodies or other biorecognition elements for the detection of the S antigen of SARS-CoV-2 (Table S3, Supporting Information). Overall, these results demonstrate the potential of aptamers for the sensitive detection of viral antigens even under non-native conditions such as VTM that would allow the analysis of clinical samples such as NP swab samples from suspected COVID-19 patients.

Application of the Assay to Clinical Samples

The assay was finally employed for the analysis of NP swab samples collected from individuals suspected of SARS-CoV-2 infection. Total RNA was extracted from these samples, and SARS-CoV-2 viral RNA was detected with droplet digital PCR (ddPCR), as described in the Supporting Information. Aptamer-based sandwich assay was performed as described in the previous section using VTM for the preparation of the standard curve samples. Ten negative samples (taken from individuals screened for COVID-19 and found not to have the infection) were analyzed, and the average signal obtained using these negative samples corresponded to 173 ng/mL, the highest signal obtained corresponded to 362 ng/mL, and 400 ng/mL was thus selected as the cutoff. Fifty samples taken from individuals who were confirmed to have the COVID-19 infection were analyzed. As can be seen in Table 2, the S1 antigen was detected in all positive samples, but applying the cutoff of 400 ng/mL established using the negative samples, nine false-negatives were obtained. Four false-positives were obtained, but these were samples taken from individuals who had been infected with COVID but in follow-up studies had then tested negative in PCR, and in these cases, there could still be some remaining antigen that was detected.
Table 2. Analysis of S1 in NP Swab Samples with the Aptamer Sandwich Assay and Comparison with PCR Results
test (target)resultnumber of samples
PCR (viral RNA)positive45
 negative5
aptamer sandwich assay (S antigen)true positive36
 false-positive3
 true negative1
 false-negative9

Conclusions

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Herein, we describe the characterization of RBD-binding aptamers for the development of a laboratory-based sandwich assay. The affinity of the aptamer candidates was evaluated, and KD values in the low nanomolar range were obtained. The specificity of the aptamers was also studied using S1 from all seven known HCoV’s, and Apt1 and Apt5 were observed to be the most specific aptamers, exhibiting only negligible cross-reactivity with the S1 from MERS-CoV. Using the Apt1 as a capture aptamer and the Apt5 as a reporter aptamer, an LOD of 21 ng/mL (270 pM) was obtained, corresponding to approximately 1 ng of S1 protein, while as low as 375 pg of S1 could be detected in VTM typically used for the collection of NP swab samples. NP swab samples from confirmed COVID-19 patients were analyzed, and the S antigen was detected in all positive samples, which had been previously verified by RT-ddPCR. However, applying the cutoff of 400 ng/mL established using the negative samples resulted in nine false-negatives. Work is ongoing to see if a component of the transport medium cross-reacts with the aptamers, resulting in this background signal, which would not be present if the swab was directly placed in a buffer, as would be the final format of the assay in a true application. In parallel, we are pursuing means to improve the assay sensitivity, combining with an antibody and also carrying out further truncation studies, and a higher number of real positive and negative samples need to be analyzed to elucidate true sensitivity and specificity values. To the best of our knowledge, this is the first report on the use of aptamers for the development of a SARS-CoV-2 antigen assay based on the S antigen. The work described herein is the first step toward the development of a rapid aptamer-based antigen test.

Experimental Section

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Materials

Phosphate-buffered saline (PBS; 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl, pH 7.4), PBS-tween (PBS, 0.05% v/v Tween 20, pH 7.4), carbonate-bicarbonate buffer (50 mM, pH 9.4), MaxiSorp immunoassay plates, and maleimide-activated plates were purchased from Fisher Scientific (Madrid, Spain). Skim milk powder was from Merck (Barcelona, Spain), BioFX TMB super-sensitive one-component HRP microwell substrate was from Surmodics (USA), streptavidin-polyHRP80 (SA-polyHRP) was from Bionova Cientifica (Madrid, Spain), and VTM was from Vircell (Granada, Spain). Spike glycoprotein S1 proteins from the known HCoV’s (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-HKU1, HCoV-OC43, and HCoV-229E, Table S1, Supporting Information) and the spike RBD from SARS-CoV-2 were supplied from Sino Biological (Eschborn, Germany). The DNA aptamers, (46) with 5′-biotin or 5′-thiol-C6 modifications, were synthesized by Biomers.net (Ulm, Germany), and their sequences are shown in Table S2. The aptamer-binding buffer (BB) was composed of PBS containing 0.55 mM MgCl2.

Evaluation of the Binding Properties of the Aptamers with a Direct Enzyme-Linked Aptamer Assay

The affinity and specificity of the aptamers were evaluated with a direct ELAA. For the affinity studies, SARS-CoV-2 spike S1 or RBD proteins (50 μL of 2 μg/mL in 50 mM carbonate buffer pH 9.4) were used to coat the wells of a 96-well plate, followed by blocking with 200 μL of 5% skimmed milk (w/v) in PBST. The wells were then incubated with 50 μL of serially diluted biotin-modified aptamers in binding buffer (200 nM down to 12.8 pM), followed by the addition of 50 μL of SA-polyHRP (50 ng/mL in PBST) for detection. Finally, 50 μL of the TMB substrate was added to each well and color development proceeded for 5 min at which point the reaction was stopped via the addition of an equal volume of 1 M sulfuric acid. The absorbance was read at 450 nm using a SpectraMax 340 PC microtiter plate reader. The affinity dissociation constants (KD values) were determined by nonlinear regression analysis performed with the GraphPad Prism 6 software using the “one site-specific binding with Hill slope” model. For the specificity studies, S1 proteins from all seven known HCoV’s (2 μg/mL each of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-HKU1, HCoV-229E, and HCoV-OC43) were immobilized on the plates and used in combination with 200 nM biotinylated aptamers as described above. All steps were performed for 30 min at ambient temperature (22–25 °C) under mild shaking conditions and the plates were washed (3 × 200 μL of PBST) between incubation steps. The relative binding (%) of the individual aptamers to each protein was calculated after normalization of the signals obtained to the responses for the target S1 protein from SARS-CoV-2. Data shown represent the mean ± SD from duplicate measurements and three independent experiments.

Screening of Aptamer Pairs for Sandwich ELAA Development

Sandwich-type ELAA assay was developed based on thiol-modified aptamers immobilized on maleimide-activated microtiter plate wells as capture aptamers in combination with biotinylated aptamers as reporters and SA-polyHRP for final detection. For screening of aptamer pairs and identification of aptamers forming a sandwich, each thiolated aptamer was used to coat the plate wells (50 μL of 500 nM in PBS) after overnight incubation at 4 °C. The plates were blocked with 200 μL of 5% skimmed milk (w/v) in PBST for 1 h at ambient temperature and then 50 μL of 2 μg/mL SARS-CoV-2 S1 protein in binding buffer were added to the wells. Following another 30 min incubation at ambient temperature and washing, the biotinylated aptamer (50 μL of 200 nM each in binding buffer) and SA-polyHRP (50 μL of 50 ng/mL in PBST) were added in sequential incubation steps followed by signal generation using TMB as described in the previous section. In total, 48 different aptamer combinations were evaluated for the detection of SARS-CoV-2 S1 in pairwise combinatorial experiments. The sensitivity of eight aptamer pairs was then studied. The pairs chosen were Apt1T/Apt5B, Apt2T/Apt1B, Apt3T/Apt1B, Apt4T/Apt1B, Apt5T/Apt1B, Apt6T/Apt6B, Apt1CT/Apt6B, and Apt4CT/Apt6B. Solutions of different concentrations of SARS-CoV-2 S1 protein were analyzed in the range of 42 pM–654 nM (equivalent to 3.2 ng/mL–50 μg/mL) using the conditions described above. The data were plotted using GraphPad Prism 6 software and fitted to a “Sigmoidal 4 parameter logistic” model. The LODs were interpolated from the fitted curves as the bottom of the curves plus three times the standard deviation of the bottom. The specificity of the same aptamer combinations was finally assessed using S1 proteins (2 μg/mL each in binding buffer) from all seven HCoV’s as described above. The data represent the mean ± SD from duplicate measurements and three independent experiments.

Optimization of Sandwich Assay Conditions for S1 Detection with the Apt1T/Apt5B Aptamer Pair

A checkerboard titration experiment was initially performed, varying the concentrations of both capture (Apt1T) and detection (Apt5B) aptamers, to optimize their concentrations at a constant SARS-CoV-2 S1 protein concentration (2 μg/mL). Apt1T starting solution of 2000 nM was serially diluted twofold down to 31.3 nM, whereas Apt5B was used in the range of 250 nM down to 7.8 nM. Next, the performance of the assay was evaluated to elucidate the LOD and EC50 using the Apt1T at the maximum concentration (2000 nM) to coat the maleimide-activated plate and the Apt5B at 125, 250, and 500 nM. S1 protein was analyzed in the range of 50 μg/mL down to 3.2 ng/mL using 50 μL per well, as detailed in the previous section.

Detection of the S Antigen in NP Swab Samples

The sandwich aptamer assay with the Apt1T/Apt5B aptamer pair was finally employed to analyze NP swab samples from the COVID-19 Cohort of the Galicia Sur Health Research Institute (COHVID-GS) (https://www.iisgaliciasur.es/apoyo-a-la-investigacion/cohorte-covid19/) including samples from individuals with a confirmed diagnosis of COVID-19 (n = 50) and healthy donors (n = 10). The specimens were collected in VTM, and ddPCR was performed to verify the presence of viral RNA in the samples, as detailed in the Supporting Information The samples were heated for 30 min at 65 °C to deactivate any remaining viral particles and used without dilution for analysis, as explained in the previous sections with some modifications. Specifically, 500 nM Apt1T capture aptamer was immobilized on the wells of the maleimide-activated plate, the volume of the sample or the standard protein was 25 μL instead of 50 μL, the sample incubation step was 1 h instead of 30 min, and the Apt5B reporter aptamer was used at 150 nM. The S1 protein standard calibration curve was constructed in parallel in VTM to facilitate the quantification of the antigen level in the samples. Each sample was analyzed in duplicate and the average signals obtained for each sample in wells with and without the Apt1T aptamer were used to interpolate the S1 antigen concentration from the calibration curve.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c05521.

  • Recombinant proteins and aptamers used in this work; biosensors and assays developed for the detection of the SARS-CoV-2 S antigen; binding studies by direct ELAA to monitor S proteins interactions with aptamers; checkerboard titration of concentrations of capture and detection aptamers to optimize aptamer sandwich assay; sensitivity of the sandwich aptamer assay at different concentrations of reporter aptamer; and SARS-CoV-2 RNA detection in NP swab samples by droplet digital PCR analysis (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
    • Ciara K. O’Sullivan - INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain;  Institució Catalana de Recerca i Estudis Avancats (ICREA), Passeig Lluís Companys 23, Barcelona 08010, Spain;  Orcidhttps://orcid.org/0000-0003-2603-2230;  Email: [email protected]
  • Authors
    • Marketa Svobodova - INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
    • Vasso Skouridou - INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain;  Orcidhttps://orcid.org/0000-0002-9712-5429
    • Miriam Jauset-Rubio - INTERFIBIO Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona 43007, Spain
    • Irene Viéitez - Rare Diseases & Pediatric Medicine Research Group, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-Uvigo, Vigo 36213, Spain;  Orcidhttps://orcid.org/0000-0001-9241-0207
    • Alberto Fernández-Villar - Pneumology Service, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-Uvigo, Vigo 36213, Spain
    • Jorge Julio Cabrera Alvargonzalez - Microbiology Service, Galicia Sur Health Research Institute (IIS Galicia Sur). SERGAS-UVigo, Vigo 36213, Spain
    • Eva Poveda - Group of Virology and Pathogenesis, Galicia Sur Health Research Institute (IIS Galicia Sur)-Complexo Hospitalario Universitario de Vigo, SERGAS-UVigo, Vigo 36213, Spain
    • Clara Benavent Bofill - Laboratori Clinic ICS Camp de Tarragona, Hospital Universitari de Tarragona Joan XXIII, Avda. Dr. Mallafré Guasch, 4, Tarragona 43007, Spain
    • Teresa Sans - Laboratori Clinic ICS Camp de Tarragona, Hospital Universitari de Tarragona Joan XXIII, Avda. Dr. Mallafré Guasch, 4, Tarragona 43007, Spain
    • Abdulaziz Bashammakh - Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 80215, Kingdom of Saudi Arabia
    • Abdulrahman O. Alyoubi - Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 80215, Kingdom of Saudi Arabia
  • Author Contributions

    M.S. and V.S. contributed equally.

  • Funding

    The COHVID-GS is funded by Fondo COVID-19 of Instituto de Salud Carlos III (COV20/00698) and Fundación Biomédica Galicia Sur. King Abdulaziz University has contributed to the funding of this work.

  • Notes
    The authors declare no competing financial interest.

    On behalf of the Cohort COVID-19 of the Galicia Sur Health Research Institute.

Acknowledgments

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We would like to thank all the members of COHVID-GS and IISGS Biobank. Members of COHVID-GS (Galicia Sur Health Research Institute): Alejandro Araujo, Jorge Julio Cabrera, Víctor del Campo, Manuel Crespo, Alberto Fernández, Beatriz Gil de Araujo, Carlos Gómez, Virginia Leiro, María Rebeca Longueira, Ana López-Domínguez, José Ramón Lorenzo, María Marcos, Alexandre Pérez, María Teresa Pérez, Lucia Patiño, Sonia Pérez, Silvia Pérez-Fernández, E.P., Cristina Ramos, Benito Regueiro, Cristina Retresas, Tania Rivera, Olga Souto, Isabel Taboada, Susana Teijeira, María Torres, Vanesa Val, and I.V.é

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  11. 11
    Liu, S.; Xiao, G.; Chen, Y.; He, Y.; Niu, J.; Escalante, C. R.; Xiong, H.; Farmar, J.; Debnath, A. K.; Tien, P.; Jiang, S. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 2004, 363, 938– 947,  DOI: 10.1016/s0140-6736(04)15788-7
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    11
    Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors
    Liu, Shuwen; Xiao, Gengfu; Chen, Yibang; He, Yuxian; Niu, Jinkui; Escalante, Carlos R.; Xiong, Huabao; Farmar, James; Debnath, Asim K.; Tien, Po; Jiang, Shibo
    Lancet (2004), 363 (9413), 938-947CODEN: LANCAO; ISSN:0140-6736. (Elsevier Science Ltd.)
    Studies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-assocd. coronavirus (SARS-CoV). We tested the inhibitory activity against infection of two sets of peptides corresponding to sequences of SARS-CoV spike protein HR1 and HR2 regions and investigated the interactions between the HR1 and HR2 peptides by surface plasmon resonance, sedimentation equilibration anal., CD, native polyacrylamide-gel electrophoresis, size exclusion high-performance liq. chromatog., and computer-aided homol. modeling and mol. docking anal. One peptide, CP-1, derived from the HR2 region, inhibited SARS-CoV infection in the micromolar range. CP-1 bound with high affinity to a peptide from the HR1 region, NP-1. CP-1 alone had low α-helicity and self-assocd. to form a trimer in phosphate buffer (pH 7.2). CP-1 and NP-1 mixed in equimolar concns. formed a six-helix bundle, similar to the fusogenic core structure of HIV-1 gp41. After binding to the target cell, the transmembrane spike protein might change conformation by assocn. between the HR1 and HR2 regions to form an oligomeric structure, leading to fusion between the viral and target-cell membranes. At the pre-fusion intermediate state, CP-1 could bind to the HR1 region and interfere with the conformational changes, resulting in inhibition of SARS-CoV fusion with the target cells. CP-1 might be modifiable to increase its anti-SARS-CoV activity and could be further developed as an antiviral agent for treatment or prophylaxis of SARS-CoV infection.
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  12. 12
    Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell. Mol. Immunol. 2020, 17, 613– 620,  DOI: 10.1038/s41423-020-0400-4
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    Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine
    Tai, Wanbo; He, Lei; Zhang, Xiujuan; Pu, Jing; Voronin, Denis; Jiang, Shibo; Zhou, Yusen; Du, Lanying
    Cellular & Molecular Immunology (2020), 17 (6), 613-620CODEN: CMIEAO; ISSN:1672-7681. (Nature Research)
    The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.
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    Dai, L.; Gao, G. F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 2021, 21, 73– 82,  DOI: 10.1038/s41577-020-00480-0
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    Viral targets for vaccines against COVID-19
    Dai, Lianpan; Gao, George F.
    Nature Reviews Immunology (2021), 21 (2), 73-82CODEN: NRIABX; ISSN:1474-1733. (Nature Research)
    A review of the progress of which viral elements are used in COVID-19 vaccine candidates, why they might act as good targets for the immune system and the implications for protective immunity. Vaccines are urgently needed to control the coronavirus disease 2019 (COVID-19) pandemic and to help the return to pre-pandemic normalcy. A great many vaccine candidates are being developed, several of which have completed late-stage clin. trials and are reporting pos. results.
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  14. 14
    Khan, S.; Tombuloglu, H.; Hassanein, S. E.; Rehman, S.; Bozkurt, A.; Cevik, E.; Abdel-Ghany, S.; Nabi, G.; Ali, A.; Sabit, H. Coronavirus diseases 2019: current biological situation and potential therapeutic perspective. Eur. J. Pharmacol. 2020, 886, 1733447,  DOI: 10.1016/j.ejphar.2020.173447
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    Tombuloglu, H.; Sabit, H.; Al-Suhaimi, E.; Al Jindan, R.; Alkharsah, K. R. Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2. PLoS One 2021, 16, e0250942  DOI: 10.1371/journal.pone.0250942
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    Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2
    Tombuloglu, Huseyin; Sabit, Hussein; Al-Suhaimi, Ebtesam; Al Jindan, Reem; Alkharsah, Khaled R.
    PLoS One (2021), 16 (4), e0250942CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-neg. and false-pos. results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clin. SARS-CoV-2 pos. samples. The results are in good agreement with the CDC authorized Cepheid's Xpert Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.
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    Ravi, N.; Cortade, D. L.; Ng, E.; Wang, S. X. Diagnostics for SARS-CoV-2 detection: a comprehensive review of the FDA-EUA COVID-19 testing landscape. Biosens. Bioelectron. 2020, 165, 112454,  DOI: 10.1016/j.bios.2020.112454
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    Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape
    Ravi, Neeraja; Cortade, Dana L.; Ng, Elaine; Wang, Shan X.
    Biosensors & Bioelectronics (2020), 165 (), 112454CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    A review. The rapidly spreading outbreak of COVID-19 disease is caused by the SARS-CoV-2 virus, first reported in Dec. 2019 in Wuhan, China. As of June 17, 2020, this virus has infected over 8.2 million people but ranges in symptom severity, making it difficult to assess its overall infection rate. There is a need for rapid and accurate diagnostics to better monitor and prevent the spread of COVID-19. In this review, we present and evaluate two main types of diagnostics with FDA-EUA status for COVID-19: nucleic acid testing for detection of SARS-CoV-2 RNA, and serol. assays for detection of SARS-CoV-2 specific IgG and IgM patient antibodies, along with the necessary sample prepn. for accurate diagnoses. In particular, we cover and compare tests such as the CDC 2019-nCoV RT-PCR Diagnostic Panel, Cellex's qSARS-CoV-2 IgG/IgM Rapid Test, and point-of-care tests such as Abbott's ID NOW COVID-19 Test. Antibody testing is esp. important in understanding the prevalence of the virus in the community and to identify those who have gained immunity. We conclude by highlighting the future of COVID-19 diagnostics, which include the need for quant. testing and the development of emerging biosensors as point-of-care tests.
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    Abbott Diagnostics Scarborough Inc. BinaxNOWTM COVID-19 Ag CARD [package insert, EUA]; Scarborough: MA, USA, 2020.
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    LumiraDx Group Limited. LumiraDxTM SARS-CoV-2 Ag Test [package insert, EUA]; Dumyat Business Park Alloa FK10 2PB: U.K., 2020.
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    Becton, Dickinson and Company. BD VeritorTM System for Rapid Detection of SARS-CoV-2 [package insert, EUA]; Sparks-Glencoe: MD, USA, 2020.
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    Quidel Corporation. Sofia Severe Acute Respiratory Syndrome Antigen FIA [package insert, EUA]: San Diego, CA, USA, 2020.
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    There is no corresponding record for this reference.
  21. 21
    Barlev-Gross, M.; Weiss, S.; Ben-Shmuel, A.; Sittner, A.; Eden, K.; Mazuz, N.; Glinert, I.; Bar-David, E.; Puni, R.; Amit, S.; Kriger, O.; Schuster, O.; Alcalay, R.; Makdasi, E.; Epstein, E.; Noy-Porat, T.; Rosenfeld, R.; Achdout, H.; Mazor, O.; Israely, T.; Levy, H.; Mechaly, A. Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens. Anal. Bioanal. Chem. 2021, 413, 3501– 3510,  DOI: 10.1007/s00216-021-03298-4
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    21
    Spike vs. nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens
    Barlev-Gross, Moria; Weiss, Shay; Ben-Shmuel, Amir; Sittner, Assa; Eden, Keren; Mazuz, Noam; Glinert, Itai; Bar-David, Elad; Puni, Reut; Amit, Sharon; Kriger, Or; Schuster, Ofir; Alcalay, Ron; Makdasi, Efi; Epstein, Eyal; Noy-Porat, Tal; Rosenfeld, Ronit; Achdout, Hagit; Mazor, Ohad; Israely, Tomer; Levy, Haim; Mechaly, Adva
    Analytical and Bioanalytical Chemistry (2021), 413 (13), 3501-3510CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many antigen detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false pos. results. We developed a novel alternative spike-based antigen assay, comprising 4 high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an inhouse nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR pos. and neg. nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clin. setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs. specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results.
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  22. 22
    Aoki, K.; Nagasawa, T.; Ishii, Y.; Yagi, S.; Okuma, S.; Kashiwagi, K.; Maeda, T.; Miyazaki, T.; Yoshizawa, S.; Tateda, K. Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs. J. Infect. Chemother. 2021, 27, 613– 616,  DOI: 10.1016/j.jiac.2020.11.021
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    22
    Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs
    Aoki, Kotaro; Nagasawa, Tatsuya; Ishii, Yoshikazu; Yagi, Shintaro; Okuma, Sadatsugu; Kashiwagi, Katsuhito; Maeda, Tadashi; Miyazaki, Taito; Yoshizawa, Sadako; Tateda, Kazuhiro
    Journal of Infection and Chemotherapy (2021), 27 (4), 613-616CODEN: JICHFN; ISSN:1341-321X. (Elsevier B.V.)
    Expansion of the testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important issue to mitigate the pandemic of coronavirus disease-2019 (COVID-19) caused by this virus. Recently, a sensitive quant. antigen test (SQT), Lumipulse SARS-CoV-2 Ag, was developed. It is a fully automated chemiluminescent enzyme immunoassay system for SARS-CoV-2. In this study, the anal. performance of SQT was examd. using clin. specimens from nasopharyngeal swabs using reverse transcription polymerase chain reaction (RT-PCR) as a control. Receiver operating characteristic anal. of 24 SARS-CoV-2-pos. and 524 -neg. patients showed an area under the curve of 0.957 ± 0.063. Using a cut-off value of 1.34 pg/mL, the sensitivity was 91.7%, the specificity was 98.5%, and the overall rate of agreement was 98.2%. In the distribution of neg. cases, the 99.5 percentile value was 1.03 pg/mL. There was a high correlation between the viral load calcd. using the cycle threshold value of RT-PCR and the concn. of antigen. The tendency for the antigen concn. to decrease with time after disease onset correlated with that of the viral load. Presented results indicate that SQT is highly concordant with RT-PCR and should be useful for the diagnosis of COVID-19 in any clin. setting. Therefore, this fully automated kit will contribute to the expansion of the testing capability for SARS-CoV-2.
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  23. 23
    Hirotsu, Y.; Maejima, M.; Shibusawa, M.; Nagakubo, Y.; Hosaka, K.; Amemiya, K.; Sueki, H.; Hayakawa, M.; Mochizuki, H.; Tsutsui, T.; Kakizaki, Y.; Miyashita, Y.; Yagi, S.; Kojima, S.; Omata, M. Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients. Int. J. Infect. Dis. 2020, 99, 397– 402,  DOI: 10.1016/j.ijid.2020.08.029
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    23
    Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients
    Hirotsu, Yosuke; Maejima, Makoto; Shibusawa, Masahiro; Nagakubo, Yuki; Hosaka, Kazuhiro; Amemiya, Kenji; Sueki, Hitomi; Hayakawa, Miyoko; Mochizuki, Hitoshi; Tsutsui, Toshiharu; Kakizaki, Yumiko; Miyashita, Yoshihiro; Yagi, Shintaro; Kojima, Satoshi; Omata, Masao
    International Journal of Infectious Diseases (2020), 99 (), 397-402CODEN: IJIDF3; ISSN:1201-9712. (Elsevier Ltd.)
    In routine clin. practice, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is detd. by reverse-transcription PCR (RT-PCR). In the current pandemic, a more rapid and high-throughput method is in growing demand. Here, the authors validated the performance of a new antigen test (LUMIPULSE) based on chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients and 231 individual samples from 4 infected patients and 215 uninfected individuals) were analyzed for SARS-CoV-2 with quant. RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. The authors detd. the cutoff value for antigen detection using receiver operating characteristic curve anal. and compared the performance of the antigen test with that of RT-qPCR. The authors also compared the viral loads and antigen levels in serial samples from seven infected patients. Using RT-qPCR as the ref., the antigen test exhibited 55.2% sensitivity and 99.6% specificity, with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, resp. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline, along with viral clearance. This gradual decline was in contrast with the abrupt pos.-to-neg. and neg.-to-pos. status changes obsd. with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients.
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  24. 24
    Pollock, N. R.; Savage, T. J.; Wardell, H.; Lee, R.; Mathew, A.; Stengelin, M.; Sigal, G. B. Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay. J. Clin. Microbiol. 2021, 59, e03077– 20,  DOI: 10.1128/JCM.03077-20
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    24
    Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay
    Pollock, Nira R.; Savage, Timothy J.; Wardell, Hanna; Lee, Rose A.; Mathew, Anu; Stengelin, Martin; Sigal, George B.
    Journal of Clinical Microbiology (2021), 59 (4), e03077CODEN: JCMIDW; ISSN:1098-660X. (American Society for Microbiology)
    Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (CT) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clin. nasopharyngeal (NP) samples is unknown. An ultrasensitive, quant. electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD S-PLEX SARS-CoV-2 N assay) was used to measure Ag in clin. NP samples from adults and children previously tested by PCR. The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/mL and a cutoff of 0.32 pg/mL. Ag concns. measured in clin. NP samples (collected in 3.0 mL of media) ranged from less than 160 fg/mL to 2.7μg/mL. Log-transformed Ag concns. correlated tightly with CT values. In 35 adult and 101 pediatric PCR-pos. samples, the sensitivities were 91% (95% confidence interval, 77 to 98%) and 79% (70 to 87%), resp. In samples with a CT of ≤35, the sensitivities were 100% (88 to 100%) and 96% (88 to 99%), resp. In 50 adult and 40 pediatric PCR-neg. specimens, the specificities were 100% (93 to 100%) and 98% (87 to 100%), resp. Nucleocapsid concns. in clin. NP samples span 8 orders of magnitude and correlate closely with RNA concns. (CT values). The S-PLEX Ag assay showed 96 to 100% sensitivity in samples from children and adults with CT values of ≤35, and a specificity of 98 to 100%. These results clarify Ag concn. distributions in clin. samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.
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  25. 25
    Pekosz, A.; Cooper, C. K.; Parvu, V.; Li, M.; Andrews, J. C.; Manabe, Y. C.; Kodsi, S.; Leitch, J.; Gary, D. S.; Roger-Dalbert, C. Antigen-based testing but not real-time polymerase chain reaction correlates with Severe Acute Respiratory Syndrome Coronavirus 2 viral culture. Clin. Infect. Dis. 2021, 73, e2861– e2866,  DOI: 10.1093/cid/ciaa1706
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    25
    Antigen-based testing but not real-time polymerase chain reaction correlates with severe acute respiratory syndrome Coronavirus 2 viral culture
    Pekosz, Andrew; Parvu, Valentin; Li, Maggie; Andrews, Jeffrey C.; Manabe, Yukari C.; Kodsi, Salma; Gary, Devin S.; Roger-Dalbert, Celine; Leitch, Jeffry; Cooper, Charles K.
    Clinical Infectious Diseases (2021), 73 (9), e2861-e2866CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)
    Individuals can test pos. for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by mol. assays following the resoln. of their clin. disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to be pos. early in the disease course, when there is an increased likelihood of high levels of infectious virus. Upper respiratory specimens from 251 participants with coronavirus disease 2019 symptoms (≤7 days from symptom onset) were prospectively collected and tested with a lateral flow antigen test and a real-time polymerase chain reaction (rt-PCR) assay for detection of SARS-CoV-2. Specimens from a subset of the study specimens were utilized to det. the presence of infectious virus in the VeroE6TMPRSS2 cell culture model. The antigen test demonstrated a higher pos. predictive value (90%) than rt-PCR (70%) when compared to culture-pos. results. The pos. percentage agreement for detection of infectious virus for the antigen test was similar to rt-PCR when compared to culture results. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture positivity represents a significant advancement in detg. the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. SARS-CoV-2 antigen testing can facilitate low-cost, scalable, and rapid time-to-result, while providing good risk detn. of those who are likely harboring infectious virus, compared to rt-PCR.
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    Scohy, A.; Anantharajah, A.; Bodéus, M.; Kabamba-Mukadi, B.; Verroken, A.; Rodriguez-Villalobos, H. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J. Clin. Virol. 2020, 129, 104455,  DOI: 10.1016/j.jcv.2020.104455
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    26
    Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis
    Scohy, Anais; Anantharajah, Ahalieyah; Bodeus, Monique; Kabamba-Mukadi, Benoit; Verroken, Alexia; Rodriguez-Villalobos, Hector
    Journal of Clinical Virology (2020), 129 (), 104455CODEN: JCVIFB; ISSN:1386-6532. (Elsevier B.V.)
    Ensuring accurate diagnosis is essential to limit the spread of SARS-CoV-2 and for the clin. management of COVID-19. Although real-time reverse transcription polymerase chain reaction (RT- qPCR) is the current recommended lab. method to diagnose SARS-CoV-2 acute infection, several factors such as requirement of special equipment and skilled staff limit the use of these time-consuming mol. techniques. Recently, several easy to perform rapid antigen detection tests were developed and recommended in some countries as the first line of diagnostic. The aim of this study was to evaluate the performances of the Coris COVID-19 Ag Respi-Strip test, a rapid immunochromatog. test for the detection of SARS-CoV-2 antigen, in comparison to RT-qPCR. 148 Nasopharyngeal swabs were tested. Amongst the 106 pos. RT-qPCR samples, 32 were detected by the rapid antigen test, given an overall sensitivity of 30.2%. All the samples detected pos. with the antigen rapid test were also pos. with RT-qPCR. Higher viral loads are assocd. with better antigen detection rates. Unfortunately, the overall poor sensitivity of the COVID-19 Ag Respi-Strip does not allow using it alone as the frontline testing for COVID-19 diagnosis.
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    Tang, Y. W.; Schmitz, J. E.; Persing, D. H.; Stratton, C. W. Laboratory diagnosis of COVID-19: current issues and challenges. J. Clin. Microbiol. 2020, 58, 10– 1128,  DOI: 10.1128/JCM.00512-20
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  28. 28
    Che, X.-y.; Qiu, L.-w.; Pan, Y.-x.; Wen, K.; Hao, W.; Zhang, L.-y.; Wang, Y.-d.; Liao, Z.-y.; Hua, X.; Cheng, V. C. C.; Yuen, K.-y. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with Severe Acute Respiratory Syndrome. J. Clin. Microbiol. 2004, 42, 2629– 2635,  DOI: 10.1128/jcm.42.6.2629-2635.2004
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    28
    Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome
    Che, Xiao-yan; Qiu, Li-wen; Pan, Yu-xian; Wen, Kun; Hao, Wei; Zhang, Li-ya; Wang, Ya-di; Liao, Zhi-yong; Hua, Xu; Cheng, Vincent C. C.; Yuen, Kwok-yung
    Journal of Clinical Microbiology (2004), 42 (6), 2629-2635CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)
    A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-assocd. coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich ELISA for the detection of SARS-CoV. The assay employed a mixt. of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clin. probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a std. to establish a detection sensitivity of approximated 50 pg/mL. The linear range of detection in clin. specimens was from 100 pg/mL to 3.2 ng/mL. Using a panel of sera collected at different points in time, the amt. of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serol. confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quant. antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.
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  29. 29
    Diao, B.; Wen, K.; Chen, J.; Liu, Y.; Yuan, Z.; Han, C.; Chen, J.; Pan, Y.; Chen, L.; Dan, Y.; Wang, J.; Chen, Y.; Deng, G.; Zhou, H.; Wu, Y. Diagnosis of acute respiratory syndrome coronavirus 2 infection by detection of nucleocapsid protein. medRxiv 2020, 20032524,  DOI: 10.1101/2020.03.07.20032524
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    Grant, B. D.; Anderson, C. E.; Williford, J. R.; Alonzo, L. F.; Glukhova, V. A.; Boyle, D. S.; Weigl, B. H.; Nichols, K. P. SARS-CoV-2 coronavirus nucleocapsid antigen-detecting half-strip lateral flow assay toward the development of point of care tests using commercially available reagents. Anal. Chem. 2020, 92, 11305– 11309,  DOI: 10.1021/acs.analchem.0c01975
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    30
    SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents
    Grant, Benjamin D.; Anderson, Caitlin E.; Williford, John R.; Alonzo, Luis F.; Glukhova, Veronika A.; Boyle, David S.; Weigl, Bernhard H.; Nichols, Kevin P.
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (16), 11305-11309CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clin. sensitive, may fill many of the gaps in the current RT-PCR testing regime, esp. in low- and middle-income countries (LMICs). To date, many serol. LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clin. sensitivity than serol. assays. To date, only a very small no. of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using com. available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equiv. to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.
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  31. 31
    Ahmadivand, A.; Gerislioglu, B.; Ramezani, Z.; Kaushik, A.; Manickam, P.; Ghoreishi, S. A. Functionalized terahertz plasmonic metasensors: femtomolar-level detection of SARS-CoV-2 spike proteins. Biosens. Bioelectron. 2021, 177, 112971,  DOI: 10.1016/j.bios.2021.112971
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    31
    Functionalized terahertz plasmonic metasensors: Femtomolar-level detection of SARS-CoV-2 spike proteins
    Ahmadivand, Arash; Gerislioglu, Burak; Ramezani, Zeinab; Kaushik, Ajeet; Manickam, Pandiaraj; Ghoreishi, S. Amir
    Biosensors & Bioelectronics (2021), 177 (), 112971CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    Effective and efficient management of human betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 virus infection i.e., COVID-19 pandemic, required sensitive and selective sensors with short sample-to-result durations for performing desired diagnostics. In this direction, one appropriate alternative approach to detect SARS-CoV-2 virus protein at low level i.e., femtomolar (fM) is exploring plasmonic metasensor technol. for COVID-19 diagnostics, which offers exquisite opportunities in advanced healthcare programs, and modern clin. diagnostics. The intrinsic merits of plasmonic metasensors stem from their capability to squeeze electromagnetic fields, simultaneously in frequency, time, and space. However, the detection of low-mol. wt. biomols. at low densities is a typical drawback of conventional metasensors that has recently been addressed using toroidal metasurface technol. This research is focused on the fabrication of a miniaturized plasmonic immunosensor based on toroidal electrodynamics concept that can sustain robustly confined plasmonic modes with ultranarrow lineshapes in the terahertz (THz) frequencies. By exciting toroidal dipole mode using our quasi-infinite metasurface and a judiciously optimized protocol based on functionalized gold nanoparticles (AuNPs) conjugated with the specific monoclonal antibody specific to spike protein (S1) of SARS-CoV-2 virus onto the metasurface, the resonance shifts for diverse concns. of the spike protein are monitored. Possessing mol. wt. around ∼76 kDa allowed to detect the presence of SARS-CoV-2 virus protein with significantly low as limit of detection (LoD) was achieved as ∼4.2 fM. We envisage that outcomes of this research will pave the way toward the use of toroidal metasensors as practical technologies for rapid and precise screening of SARS-CoV-2 virus carriers, symptomatic or asymptomatic, and spike proteins in hospitals, clinics, labs., and site of infection.
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  32. 32
    Seo, G.; Lee, G.; Kim, M. J.; Baek, S.-H.; Choi, M.; Ku, K. B.; Lee, C.-S.; Jun, S.; Park, D.; Kim, H. G.; Kim, S.-J.; Lee, J.-O.; Kim, B. T.; Park, E. C.; Kim, S. I. Rapid detection of COVID-19 causative virus (SARS-CoV-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor. ACS Nano 2020, 14, 5135– 5142,  DOI: 10.1021/acsnano.0c02823
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    32
    Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor
    Seo, Giwan; Lee, Geonhee; Kim, Mi Jeong; Baek, Seung-Hwa; Choi, Minsuk; Ku, Keun Bon; Lee, Chang-Seop; Jun, Sangmi; Park, Daeui; Kim, Hong Gi; Kim, Seong-Jun; Lee, Jeong-O.; Kim, Bum Tae; Park, Edmond Changkyun; Kim, Seung Il
    ACS Nano (2020), 14 (4), 5135-5142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for contg. the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clin. samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was detd. using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concns. of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clin. transport medium. In addn., the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 x 101 pfu/mL) and clin. samples (LOD: 2.42 x 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunol. diagnostic method for COVID-19 that requires no sample pretreatment or labeling.
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  33. 33
    Zhong, J.; Rösch, E. L.; Viereck, T.; Schilling, M.; Ludwig, F. Toward rapid and sensitive detection of SARS-CoV-2 with functionalized magnetic nanoparticles. ACS Sens. 2021, 6, 976– 984,  DOI: 10.1021/acssensors.0c02160
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    33
    Toward Rapid and Sensitive Detection of SARS-CoV-2 with Functionalized Magnetic Nanoparticles
    Zhong, Jing; Roesch, Enja Laureen; Viereck, Thilo; Schilling, Meinhard; Ludwig, Frank
    ACS Sensors (2021), 6 (3), 976-984CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global medical systems and economies and rules our daily living life. Controlling the outbreak of SARS-CoV-2 has become 1 of the most important and urgent strategies throughout the whole world. As of Oct. 2020, there have not yet been any medicines or therapies to be effective against SARS-CoV-2. Thus, rapid and sensitive diagnostics is the most important measures to control the outbreak of SARS-CoV-2. Homogeneous biosensing based on magnetic nanoparticles (MNPs) is 1 of the most promising approaches for rapid and highly sensitive detection of biomols. This paper proposes an approach for rapid and sensitive detection of SARS-CoV-2 with functionalized MNPs via the measurement of their magnetic response in an ac magnetic field. For proof of concept, mimic SARS-CoV-2 consisting of spike proteins and polystyrene beads are used for expts. Exptl. results demonstrate that the proposed approach allows the rapid detection of mimic SARS-CoV-2 with a limit of detection of 0.084 nM (5.9 fmol). The proposed approach has great potential for designing a low-cost and point-of-care device for rapid and sensitive diagnostics of SARS-CoV-2.
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  34. 34
    Ventura, B. D.; Cennamo, M.; Minopoli, A.; Campanile, R.; Censi, S. B.; Terracciano, D.; Portella, G.; Velotta, R. Colorimetric test for fast detection of SARS-CoV-2 in nasal and throat swabs. ACS Sens. 2020, 5, 3043– 3048,  DOI: 10.1021/acssensors.0c01742
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    34
    Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs
    Ventura, Bartolomeo Della; Cennamo, Michele; Minopoli, Antonio; Campanile, Raffaele; Censi, Sergio Bolletti; Terracciano, Daniela; Portella, Giuseppe; Velotta, Raffaele
    ACS Sensors (2020), 5 (10), 3043-3048CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biol. material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal soln. of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting 3 surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a soln. contg. the viral particle. The optical d. of the mixed soln. measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold std. for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiol., as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concn.
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  35. 35
    Pramanik, A.; Gao, Y.; Patibandla, S.; Mitra, D.; McCandless, M. G.; Fassero, L. A.; Gates, K.; Tandon, R.; Chandra Ray, P. The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles. Nanoscale Adv. 2021, 3, 1588– 1596,  DOI: 10.1039/d0na01007c
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    35
    The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles
    Pramanik, Avijit; Gao, Ye; Patibandla, Shamily; Mitra, Dipanwita; McCandless, Martin G.; Fassero, Lauren A.; Gates, Kalein; Tandon, Ritesh; Chandra Ray, Paresh
    Nanoscale Advances (2021), 3 (6), 1588-1596CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease that began in 2019 (COVID-19), has been responsible for 1.4 million deaths worldwide as of 13 Nov. 2020. Because at the time of writing no vaccine is yet available, a rapid diagnostic assay is very urgently needed. Herein, we present the development of anti-spike antibody attached gold nanoparticles for the rapid diagnosis of specific COVID-19 viral antigen or virus via a simple colorimetric change observation within a 5 min time period. For rapid and highly sensitive identification, surface enhanced Raman spectroscopy (SERS) was employed using 4-aminothiophenol as a reporter mol., which is attached to the gold nanoparticle via an Au-S bond. In the presence of COVID-19 antigen or virus particles, owing to the antigen-antibody interaction, the gold nanoparticles undergo aggregation, changing color from pink to blue, which allows for the detn. of the presence of antigen or virus very rapidly by the naked eye, even at concns. of 1 ng (ng) per mL for COVID-19 antigen and 1000 virus particles per mL for SARS-CoV-2 spike protein pseudotyped baculovirus. Importantly, the aggregated gold nanoparticles form "hot spots" to provide very strong SERS signal enhancement from anti-spike antibody and 4-aminothiophenol attached gold nanoparticles via light-matter interactions. Finite-difference time-domain (FDTD) simulation data indicate a 4-orders-of-magnitude Raman enhancement in "hot spot" positions when gold nanoparticles form aggregates. Using a portable Raman analyzer, our reported data demonstrate that our antibody and 4-aminothiophenol attached gold nanoparticle-based SERS probe has the capability to detect COVID-19 antigen even at a concn. of 4 pg (pg) per mL and virus at a concn. of 18 virus particles per mL within a 5 min time period. Using HEK293T cells, which express angiotensin-converting enzyme 2 (ACE2), by which SARS-CoV-2 enters human cells, we show that anti-spike antibody attached gold nanoparticles have the capability to inhibit infection by the virus. Our reported data show that antibody attached gold nanoparticles bind to SARS-CoV-2 spike protein, thereby inhibiting the virus from binding to cell receptors, which stops virus infection and spread. It also has the capability to destroy the lipid membrane of the virus.
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  36. 36
    Yousefi, H.; Mahmud, A.; Chang, D.; Das, J.; Gomis, S.; Chen, J. B.; Wang, H.; Been, T.; Yip, L.; Coomes, E.; Li, Z.; Mubareka, S.; McGeer, A.; Christie, N.; Gray-Owen, S.; Cochrane, A.; Rini, J. M.; Sargent, E. H.; Kelley, S. O. Detection of SARS-CoV-2 viral particles using direct, reagent-free electrochemical sensing. J. Am. Chem. Soc. 2021, 143, 1722– 1727,  DOI: 10.1021/jacs.0c10810
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    36
    Detection of SARS-CoV-2 Viral Particles Using Direct, Reagent-Free Electrochemical Sensing
    Yousefi, Hanie; Mahmud, Alam; Chang, Dingran; Das, Jagotamoy; Gomis, Surath; Chen, Jenise B.; Wang, Hansen; Been, Terek; Yip, Lily; Coomes, Eric; Li, Zhijie; Mubareka, Samira; McGeer, Allison; Christie, Natasha; Gray-Owen, Scott; Cochrane, Alan; Rini, James M.; Sargent, Edward H.; Kelley, Shana O.
    Journal of the American Chemical Society (2021), 143 (4), 1722-1727CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combating the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold std. nucleic acid-based approaches require enzymic amplification to achieve clin. relevant levels of sensitivity and are not typically used outside of a lab. setting. We report reagent-free viral sensing that directly reads out the presence of viral particles in 5 min using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a neg. charged DNA linker that also features a tethered redox probe. When a pos. potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the 1st virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its assocd. spike protein in test samples and in unprocessed patient saliva.
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  37. 37
    Lee, J.-H.; Choi, M.; Jung, Y.; Lee, S. K.; Lee, C.-S.; Kim, J.; Kim, J.; Kim, N. H.; Kim, B.-T.; Kim, H. G. A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2). Biosens. Bioelectron. 2021, 171, 112715,  DOI: 10.1016/j.bios.2020.112715
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    37
    A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)
    Lee, Jong-Hwan; Choi, Minsuk; Jung, Yujin; Lee, Sung Kyun; Lee, Chang-Seop; Kim, Jung; Kim, Jongwoo; Kim, Nam Hoon; Kim, Bum-Tae; Kim, Hong Gi
    Biosensors & Bioelectronics (2021), 171 (), 112715CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a newly emerging human infectious disease. Because no specific antiviral drugs or vaccines are available to treat COVID-19, early diagnostics, isolation, and prevention are crucial for contg. the outbreak. Mol. diagnostics using reverse transcription polymerase chain reaction (RT-PCR) are the current gold std. for detection. However, viral RNAs are much less stable during transport and storage than proteins such as antigens and antibodies. Consequently, false-neg. RT-PCR results can occur due to inadequate collection of clin. specimens or poor handling of a specimen during testing. Although antigen immunoassays are stable diagnostics for detection of past infection, infection progress, and transmission dynamics, no matched antibody pair for immunoassay of SARS-CoV-2 antigens has yet been reported. In this study, we designed and developed a novel rapid detection method for SARS-CoV-2 spike 1 (S1) protein using the SARS-CoV-2 receptor ACE2, which can form matched pairs with com. available antibodies. ACE2 and S1-mAb were paired with each other for capture and detection in a lateral flow immunoassay (LFIA) that did not cross-react with SARS-CoV Spike 1 or MERS-CoV Spike 1 protein. The SARS-CoV-2 S1 (<5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86 x 105 copies/mL in the clin. specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects. This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.
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  38. 38
    Pinals, R. L.; Ledesma, F.; Yang, D.; Navarro, N.; Jeong, S.; Pak, J. E.; Kuo, L.; Chuang, Y.-C.; Cheng, Y.-W.; Sun, H.-Y.; Landry, M. P. Rapid SARS-CoV-2 spike protein detection by carbon nanotube-based near-infrared nanosensors. Nano Lett. 2021, 21, 2272– 2280,  DOI: 10.1021/acs.nanolett.1c00118
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    38
    Rapid SARS-CoV-2 spike protein detection by carbon nanotube-based near-infrared nanosensors
    Pinals, Rebecca L.; Ledesma, Francis; Yang, Darwin; Navarro, Nicole; Jeong, Sanghwa; Pak, John E.; Kuo, Lili; Chuang, Yung-Chun; Cheng, Yu-Wei; Sun, Hung-Yu; Landry, Markita P.
    Nano Letters (2021), 21 (5), 2272-2280CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    To effectively track and eliminate COVID-19, it is crit. to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.
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    Baker, A. N.; Richards, S.-J.; Guy, C. S.; Congdon, T. R.; Hasan, M.; Zwetsloot, A. J.; Gallo, A.; Lewandowski, J. R.; Stansfeld, P. J.; Straube, A.; Walker, M.; Chessa, S.; Pergolizzi, G.; Dedola, S.; Field, R. A.; Gibson, M. I. The SARS-COV-2 spike protein binds sialic acids and enables rapid detection in a lateral flow point of care diagnostic device. ACS Cent. Sci. 2020, 6, 2046– 2052,  DOI: 10.1021/acscentsci.0c00855
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    39
    The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device
    Baker, Alexander N.; Richards, Sarah-Jane; Guy, Collette S.; Congdon, Thomas R.; Hasan, Muhammad; Zwetsloot, Alexander J.; Gallo, Angelo; Lewandowski, Jozef R.; Stansfeld, Phillip J.; Straube, Anne; Walker, Marc; Chessa, Simona; Pergolizzi, Giulia; Dedola, Simone; Field, Robert A.; Gibson, Matthew I.
    ACS Central Science (2020), 6 (11), 2046-2052CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N-acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform. The spike-protein from SARS-COV-2 is shown to bind sialic acids, which is exploited to assemble a lateral flow diagnostic tool, using glycans rather than antibodies, as the recognition unit.
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    Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505– 510,  DOI: 10.1126/science.2200121
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    Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase
    Tuerk, Craig; Gold, Larry
    Science (Washington, DC, United States) (1990), 249 (4968), 505-10CODEN: SCIEAS; ISSN:0036-8075.
    A novel method was developed for rapidly selecting preferred binding sequences from a population of random sequences. The method is designated systematic evolution of ligands by exponential enrichment (SELEX). High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calcd. pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding consts. of these two RNA's to T4 DNA polymerase are equiv. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high affinity ligands could conceivably be developed for any target mol.
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    In vitro selection of RNA molecules that bind specific ligands
    Ellington, Andrew D.; Szostak, Jack W.
    Nature (London, United Kingdom) (1990), 346 (6287), 818-22CODEN: NATUAS; ISSN:0028-0836.
    Subpopulations of RNA mols. that bind specifically to a variety of org. dyes have been isolated from a population of random sequence RNA mols. Roughly one in 1010 random sequence RNA mols. folds in such a way as to create a specific binding site for small ligands.
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    Robertson, D. L.; Joyce, G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 1990, 344, 467– 468,  DOI: 10.1038/344467a0
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    Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA
    Robertson, Debra L.; Joyce, Gerald F.
    Nature (London, United Kingdom) (1990), 344 (6265), 467-8CODEN: NATUAS; ISSN:0028-0836.
    The discovery of RNA enzymes has, for the 1st time, provided a single mol. that has both genetic and catalytic properties. Techniques were previously devised for the mutation, selection, and amplification of catalytic RNA, all of which can be performed rapidly in vitro. This report describes how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution expts. to be carried out in response to artificially imposed selection constraints. The subject of the investigation was the Tetrahymena ribozyme, a self-splicing group I intron derived from the large rRNA precursor of Tetrahymena thermophila that catalyzes sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. Variant forms of the enzyme were selected that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected mol. represents the discovery of the 1st RNA enzyme known to cleave single-stranded DNA specifically.
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    Nguyen, V.-T.; Seo, H. B.; Kim, B. C.; Kim, S. K.; Song, C.-S.; Gu, M. B. Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers. Biosens. Bioelectron. 2016, 86, 293– 300,  DOI: 10.1016/j.bios.2016.06.064
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    Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers
    Nguyen, Van-Thuan; Seo, Ho Bin; Kim, Byoung Chan; Kim, Sang Kyung; Song, Chang-Seon; Gu, Man Bock
    Biosensors & Bioelectronics (2016), 86 (), 293-300CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    In this research, we report highly sensitive and specific sandwich-type SPR-based biosensor for the detection H5Nx whole viruses. A few of aptamers, for the first time, were successfully screened and characterized for whole avian influenza (AI) viruses, H5Nx, by using Multi-GO-SELEX method. The affinities of the aptamers developed in this study were ranged from 8×104 to 1×104 EID50/mL, and the aptamers IF22, IF23 were found to be specific to H5N1 and H5N8, resp. In addn., some flexible aptamers IF20, IF15, and IF10 were found to bind to the H5N1 and H5N2, H5N1 and H5N8, or H5N1, H5N2, and H5N8, resp. Moreover, aptamers IF10 and IF22 were found to bind H5N1 virus simultaneously and confirmed to bind the different site of the same H5N1 whole virus. Therefore, this pair of aptamers, IF10 and IF22, were successfully applied to develop the sandwich-type SPR-based biosensor assay which is rapid, accurate for the detection of AI whole virus from H5N1-infected feces samples. The min. detectible concn. of H5N1 whole virus was found to be 200 EID50/mL with this sandwich-type detection using the aptamer pair obtained in this study. In addn., the sensitivity of this biosensor was successfully enhanced by using the signal amplification with the secondary aptamer conjugated with gold nanoparticles.
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    Park, J.-W.; Jin Lee, S.; Choi, E.-J.; Kim, J.; Song, J.-Y.; Bock Gu, M. An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening. Biosens. Bioelectron. 2014, 51, 324– 329,  DOI: 10.1016/j.bios.2013.07.052
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    An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening
    Park, Jee-Woong; Jin Lee, Su; Choi, Eun-Jin; Kim, Jaejo; Song, Jae-Young; Bock Gu, Man
    Biosensors & Bioelectronics (2014), 51 (), 324-329CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    In this study, we successfully developed a ssDNA aptamer pairs by using an advanced immobilization-free SELEX method with affinity-based selection and counter-screening process at every round. By implementing this method, two different aptamers specifically binding to bovine viral diarrhea virus type 1 (BVDV type 1) with high affinity were successfully screened. This aptamer pair was applied to ultrasensitive detection platform for BVDV type 1 in a sandwich manner. The ultrasensitive detection of BVDV type 1 using one of aptamers conjugated with gold nanoparticles was obtained in aptamer-aptamer sandwich type sensing format, with the limit of detection of 800 copies/mL, which is comparable to a real-time PCR method.
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    Seo, H. B.; Gu, M. B. Aptamer-based sandwich-type biosensors. J. Biol. Eng. 2017, 11, 11,  DOI: 10.1186/s13036-017-0054-7
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    Aptamer-based sandwich-type biosensors
    Seo, Ho Bin; Gu, Man Bock
    Journal of Biological Engineering (2017), 11 (), 11/1-11/7CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
    Sandwich-type biosensor platforms have drawn lots of attentions due to its superior features, compared to other platforms, in terms of its stable and reproducible responses and easy enhancement in the detection sensitivity. The sandwich-type assays can be developed by utilizing a pair of receptors, which bind to the different sites of the same target. In this mini-review paper, the sandwich-type biosensors using either pairs of aptamers or aptamer-antibody pairs are reviewed in terms of its targets and platforms, the schematic designs, and their anal. performance.
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    Song, Y.; Song, J.; Wei, X.; Huang, M.; Sun, M.; Zhu, L.; Lin, B.; Shen, H.; Zhu, Z.; Yang, C. Discovery of aptamers targeting the receptor-binding domain of the SARS-CoV-2 spike glycoprotein. Anal. Chem. 2020, 92, 9895– 9900,  DOI: 10.1021/acs.analchem.0c01394
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    Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Song, Yanling; Song, Jia; Wei, Xinyu; Huang, Mengjiao; Sun, Miao; Zhu, Lin; Lin, Bingqian; Shen, Haicong; Zhu, Zhi; Yang, Chaoyong
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (14), 9895-9900CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, resp. Simulated interaction modeling, along with competitive expts., suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.
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    Stanborough, T.; Given, F. M.; Koch, B.; Sheen, C. R.; Stowers-Hull, A. B.; Waterland, M. R.; Crittenden, D. L. Optical detection of CoV-SARS-2 viral proteins to sub-picomolar concentrations. ACS Omega 2021, 6, 6404– 6413,  DOI: 10.1021/acsomega.1c00008
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    47
    Optical detection of CoV-SARS-2 viral proteins to sub-picomolar concentrations
    Stanborough, Tamsyn; Given, Fiona M.; Koch, Barbara; Sheen, Campbell R.; Stowers-Hull, Andre Buzas; Waterland, Mark R.; Crittenden, Deborah L.
    ACS Omega (2021), 6 (9), 6404-6413CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample vols. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiol. relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample vols., takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concns. Control expts. performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response obsd. for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or lab. expertise and equipment.
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    Pramanik, A.; Gao, Y.; Patibandla, S.; Mitra, D.; McCandless, M. G.; Fassero, L. A.; Gates, K.; Tandon, R.; Ray, P. C. Aptamer conjugated gold nanostar-based distance-dependent nanoparticle surface energy transfer spectroscopy for ultrasensitive detection and inactivation of corona virus. J. Phys. Chem. Lett. 2021, 12, 2166– 2171,  DOI: 10.1021/acs.jpclett.0c03570
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    48
    Aptamer conjugated gold nanostar-based distance-dependent nanoparticle surface energy transfer spectroscopy for ultrasensitive detection and inactivation of corona virus
    Pramanik, Avijit; Gao, Ye; Patibandla, Shamily; Mitra, Dipanwita; McCandless, Martin G.; Fassero, Lauren A.; Gates, Kalein; Tandon, Ritesh; Ray, Paresh Chandra
    Journal of Physical Chemistry Letters (2021), 12 (8), 2166-2171CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been detd. to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.
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    Schmitz, A.; Weber, A.; Bayin, M.; Breuers, S.; Fieberg, V.; Famulok, M.; Mayer, G. A SARS-CoV-2 spike binding DNA aptamer that inhibits pseudovirus infection by an RBD-independent mechanism. Angew. Chem., Int. Ed. 2021, 60, 2– 9,  DOI: 10.1101/2020.12.23.424171
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    Yang, G.; Li, Z.; Mohammed, I.; Zhao, L.; Wei, W.; Xiao, H.; Guo, W.; Zhao, Y.; Qu, F.; Huang, Y. Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1. Signal Transduction Targeted Ther. 2021, 6, 227,  DOI: 10.1038/s41392-021-00649-6
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    Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1
    Yang, Ge; Li, Ziyue; Mohammed, Irfan; Zhao, Liping; Wei, Wei; Xiao, Haihua; Guo, Weisheng; Zhao, Yongxiang; Qu, Feng; Huang, Yuanyu
    Signal Transduction and Targeted Therapy (2021), 6 (1), 227CODEN: STTTCB; ISSN:2059-3635. (Nature Research)
    Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1.
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    Li, J.; Zhang, Z.; Gu, J.; Stacey, H. D.; Ang, J. C.; Capretta, A.; Filipe, C. D. M.; Mossman, K. L.; Balion, C.; Salena, B. J.; Yamamura, D.; Soleymani, L.; Miller, M. S.; Brennan, J. D.; Li, Y. Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library. Nucleic Acids Res. 2021, 49, 7267– 7279,  DOI: 10.1093/nar/gkab574
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    51
    Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library
    Li, Jiuxing; Zhang, Zijie; Gu, Jimmy; Stacey, Hannah D.; Ang, Jann C.; Capretta, Alfredo; Filipe, Carlos D. M.; Mossman, Karen L.; Balion, Cynthia; Salena, Bruno J.; Yamamura, Deborah; Soleymani, Leyla; Miller, Matthew S.; Brennan, John D.; Li, Yingfu
    Nucleic Acids Research (2021), 49 (13), 7267-7279CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    We performed in vitro selection expts. to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with resp. Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation expts. revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.
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    Gupta, A.; Anand, A.; Jain, N.; Goswami, S.; Anantharaj, A.; Patil, S.; Singh, R.; Kumar, A.; Shrivastava, T.; Bhatnagar, S.; Medigeshi, G. R.; Sharma, T. K.; DBT India Consortium for COVID-19 Research A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2. Mol. Ther.--Nucleic Acids 2021, 26, 321– 332,  DOI: 10.1016/j.omtn.2021.06.014
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    A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2
    Gupta, Ankit; Anand, Anjali; Jain, Neha; Goswami, Sandeep; Anantharaj, Anbalagan; Patil, Sharanabasava; Singh, Rahul; Kumar, Amit; Shrivastava, Tripti; Bhatnagar, Shinjini; Medigeshi, Guruprasad R.; Sharma, Tarun Kumar
    Molecular Therapy--Nucleic Acids (2021), 26 (), 321-332CODEN: MTAOC5; ISSN:2162-2531. (Elsevier Inc.)
    The recent SARS-CoV-2 outbreak has been declared a global health emergency. It will take years to vaccinate the whole population to protect them from this deadly virus, hence the management of SARS-CoV-2 largely depends on the widespread availability of an accurate diagnostic test. Toward addressing the unmet need of a reliable diagnostic test in the current work by utilizing the power of Systematic Evolution of Ligands by EXponential enrichment, a 44-mer G-quadruplex-forming DNA aptamer against spike trimer antigen of SARS-CoV-2 was identified. The lead aptamer candidate (S14) was characterized thoroughly for its binding, selectivity, affinity, structure, and batch-to-batch variability by utilizing various biochem., biophys., and in silico techniques. S14 has demonstrated a low nanomolar KD, confirming its tight binding to a spike antigen of SARS-CoV-2. S14 can detect as low as 2 nM of antigen. The clin. evaluation of S14 aptamer on nasopharyngeal swab specimens (n = 232) has displayed a highly discriminatory response between SARS-CoV-2 infected individuals from the non-infected one with a sensitivity and specificity of ∼91% and 98%, resp. Importantly, S14 aptamer-based test has evinced a comparable performance with that of RT-PCR-based assay. Altogether, this study established the utility of aptamer technol. for the detection of SARS-CoV-2.
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    Alves Ferreira-Bravo, I.; DeStefano, J. J. Xeno-nucleic Acid (XNA) 2’-fluoro-arabino nucleic acid (FANA) aptamers to the receptor-binding domain of SARS-CoV-2 S protein block ACE2 binding. Viruses 2021, 13, 1983,  DOI: 10.3390/v13101983
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    53
    Xeno-Nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding
    Alves Ferreira-Bravo, Irani; DeStefano, Jeffrey J.
    Viruses (2021), 13 (10), 1983CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ∼79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equil. dissocn. consts. (KD,app) of 10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, resp. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per wt. basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.
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    Behbahani, M.; Mohabatkar, H.; Hosseini, B. In silico design of quadruplex aptamers against the spike protein of SARS-CoV-2. Inf. Med. 2021, 26, 100757,  DOI: 10.1016/j.imu.2021.100757
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    In silico design of quadruplex aptamers against the spike protein of SARS-CoV-2
    Behbahani Mandana; Mohabatkar Hassan; Hosseini Barumand
    Informatics in medicine unlocked (2021), 26 (), 100757 ISSN:2352-9148.
    Nucleic acid aptamers are short sequences of nucleic acid ligands that bind to a specific target molecule. Aptamers are experimentally nominated using the well-designed SELEX (systematic evolution of ligands by exponential enrichment) method. Here, we designed a new method for diagnosis and blocking SARS-CoV-2 based on G-quadruplex aptamer. This aptamer was developed against the receptor-binding domain (RBD) region of the spike protein. In the current study, ten quadruplex DNA aptamers entitled AP1, AP2, AP3, AP4, AP5, AP6, AP7, AP8, AP9, and AP10 were designed in silico and had high HADDOCK scores. One quadruplex aptamer sequence (AP1) was selected based on the interaction with RBD of SARS-CoV-2. Results showed that AP1 aptamer could be used as an agent in the diagnosis and therapy of SARS-CoV-2, although more works are still needed.
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    Devi, A.; Chaitanya, N. S. N. Designing of peptide aptamer targeting the receptor-binding domain of spike protein of SARS-CoV-2: an in silico study. Mol. Diversity 2021, 1– 13,  DOI: 10.1007/s11030-020-10171-6
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    Artyushenko, P. V.; Mironov, V. A.; Mironov, V. A.; Morozov, D. I.; Shchugoreva, I. A.; Borbone, N.; Tomilin, F. N.; Kichkailo, A. S. Computational approach to design of aptamers to the receptor binding domain of SARS-CoV-2. Sib. Med. Rev. 2021, 2, 66– 67,  DOI: 10.20333/25000136-2021-2-66-67
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    Kowada, A. Greater public health impact of COVID-19 antigen detection tests. BMC Med. 2021, 19, 82,  DOI: 10.1186/s12916-021-01956-z
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    Greater public health impact of COVID-19 antigen detection tests
    Kowada, Akiko
    BMC Medicine (2021), 19 (1), 82CODEN: BMMECZ; ISSN:1741-7015. (BioMed Central Ltd.)
    Early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevents widespread transmission, manages outbreak control, improves poor prognosis, and reduces mortality of the Coronavirus disease 2019 (COVID-19). Ricks et al. report in BMC Medicine greater healthcare cost-saving and health outcomes of antigen detection rapid diagnostic tests compared to nucleic acid tests for symptomatic persons. The main outcomes in the study are the health system costs and health impacts (deaths averted and infectious days isolated). Antigen-detection rapid diagnostic test-led strategy is compared to a strategy based on nucleic acid tests and clin. judgment.
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  • Abstract

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    Figure 1

    Figure 1. Detection of the SARS-CoV-2 S antigen with the aptamer sandwich assay.

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    Figure 2

    Figure 2. Specificity of the aptamers evaluated by direct ELAA. The relative binding of each aptamer was calculated using constant concentrations of aptamers (200 nM) and S1 proteins from the different HCoV’s (2 μg/mL). The error bars correspond to the standard deviation from duplicate measurements.

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    Figure 3

    Figure 3. Screening of aptamer pairs for the detection of SARS-CoV-2 S1 protein. Each thiolated capture aptamer (AptxT) was analyzed in combination with each of the biotinylated reporter aptamers (AptxB) using a constant concentration of S1. No protein was added for the control samples. The number refers to the number of the aptamer tested, T indicates the thiolated aptamer and B indicates the biotinylated aptamer.

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    Figure 4

    Figure 4. Sensitivity of the sandwich assays for SARS-CoV-2 S1 detection using different combinations of aptamer pairs.

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    Figure 5

    Figure 5. Specificity of the sandwich assays using different aptamer pairs and S1 from different HCoV’s. Each protein was used at 2 μg/mL, and the error bars indicate the standard deviations from duplicate measurements.

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    Figure 6

    Figure 6. Sensitivity of the sandwich assay based on the Apt1T/Apt5B aptamer pair in aptamer binding buffer and in VTM.

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  • References

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      A review. In late Dec. 2019, a cluster of unexplained pneumonia cases has been reported in Wuhan, China. A few days later, the causative agent of this mysterious pneumonia was identified as a novel coronavirus. This causative virus has been temporarily named as severe acute respiratory syndrome coronavirus 2 and the relevant infected disease has been named as coronavirus disease 2019 (COVID-19) by the World Health Organization, resp. The COVID-19 epidemic is spreading in China and all over the world now. The purpose of this review is primarily to review the pathogen, clin. features, diagnosis, and treatment of COVID-19, but also to comment briefly on the epidemiol. and pathol. based on the current evidence.
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    3. 3
      Zhu, Z.; Lian, X.; Su, X.; Wu, W.; Marraro, G. A.; Zeng, Y. From SARS and MERS to COVID-19: a brief summary and comparison of severe acute respiratory infections caused by three highly pathogenic human coronaviruses. Respir. Res. 2020, 21, 224,  DOI: 10.1186/s12931-020-01479-w
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      3
      From SARS and MERS to COVID-19: a brief summary and comparison of severe acute respiratory infections caused by three highly pathogenic human coronaviruses
      Zhu, Zhixing; Lian, Xihua; Su, Xiaoshan; Wu, Weijing; Marraro, Giuseppe A.; Zeng, Yiming
      Respiratory Research (2020), 21 (1), 224CODEN: RREEBZ; ISSN:1465-993X. (BioMed Central Ltd.)
      A review. Within 2 decades, there have emerged 3 highly pathogenic and deadly human coronaviruses, namely SARS-CoV, MERS-CoV, and SARS-CoV-2. The economic burden and health threats caused by these coronaviruses are extremely dreadful and getting more serious as the increasing no. of global infections and attributed deaths of SARS-CoV-2 and MERS-CoV. Unfortunately, specific medical countermeasures for these hCoVs remain absent. Moreover, the fast spread of misinformation about the ongoing SARS-CoV-2 pandemic uniquely places the virus alongside an annoying infodemic and causes unnecessary worldwide panic. SARS-CoV-2 shares many similarities with SARS-CoV and MERS-CoV, certainly, obvious differences exist as well. Lessons learned from SARS-CoV and MERS-CoV, timely updated information of SARS-CoV-2 and MERS-CoV, and summarized specific knowledge of these hCoVs are extremely invaluable for effectively and efficiently contain the outbreak of SARS-CoV-2 and MERS-CoV. By gaining a deeper understanding of hCoVs and the illnesses caused by them, we can bridge knowledge gaps, provide cultural weapons for fighting and controlling the spread of MERS-CoV and SARS-CoV-2, and prep. effective and robust defense lines against hCoVs that may emerge or reemerge in the future. To this end, the state-of-the-art knowledge and comparing the biol. features of these lethal hCoVs and the clin. characteristics of illnesses caused by them are systematically summarized in the review.
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    4. 4
      Gorbalenya, A. E.; Baker, S. C.; Baric, R. S.; de Groot, R. J.; Drosten, C.; Gulyaeva, A. A.; Haagmans, B. L.; Lauber, C.; Leontovich, A. M.; Neuman, B. W.; Penzar, D.; Perlman, S.; Poon, L. L. M.; Samborskiy, D. V.; Sidorov, I. A.; Sola, I.; Ziebuhr, J. The species Severe Acute Respiratory Syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020, 5, 536– 544,  DOI: 10.1038/s41564-020-0695-z
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    5. 5
      Wrapp, D.; Wang, N.; Corbett, K. S.; Goldsmith, J. A.; Hsieh, C.-L.; Abiona, O.; Graham, B. S.; McLellan, J. S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020, 367, 1260– 1263,  DOI: 10.1126/science.abb2507
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      5
      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation
      Wrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.
      Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
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    6. 6
      Wang, N.; Shang, J.; Jiang, S.; Du, L. Subunit vaccines against emerging pathogenic human coronaviruses. Front. Microbiol. 2020, 11, 298,  DOI: 10.3389/fmicb.2020.00298
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      6
      Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses
      Wang Ning; Jiang Shibo; Du Lanying; Shang Jian; Jiang Shibo
      Frontiers in microbiology (2020), 11 (), 298 ISSN:1664-302X.
      Seven coronaviruses (CoVs) have been isolated from humans so far. Among them, three emerging pathogenic CoVs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and a newly identified CoV (2019-nCoV), once caused or continue to cause severe infections in humans, posing significant threats to global public health. SARS-CoV infection in humans (with about 10% case fatality rate) was first reported from China in 2002, while MERS-CoV infection in humans (with about 34.4% case fatality rate) was first reported from Saudi Arabia in June 2012. 2019-nCoV was first reported from China in December 2019, and is currently infecting more than 70000 people (with about 2.7% case fatality rate). Both SARS-CoV and MERS-CoV are zoonotic viruses, using bats as their natural reservoirs, and then transmitting through intermediate hosts, leading to human infections. Nevertheless, the intermediate host for 2019-nCoV is still under investigation and the vaccines against this new CoV have not been available. Although a variety of vaccines have been developed against infections of SARS-CoV and MERS-CoV, none of them has been approved for use in humans. In this review, we have described the structure and function of key proteins of emerging human CoVs, overviewed the current vaccine types to be developed against SARS-CoV and MERS-CoV, and summarized recent advances in subunit vaccines against these two pathogenic human CoVs. These subunit vaccines are introduced on the basis of full-length spike (S) protein, receptor-binding domain (RBD), non-RBD S protein fragments, and non-S structural proteins, and the potential factors affecting these subunit vaccines are also illustrated. Overall, this review will be helpful for rapid design and development of vaccines against the new 2019-nCoV and any future CoVs with pandemic potential. This review was written for the topic of Antivirals for Emerging Viruses: Vaccines and Therapeutics in the Virology section of Frontiers in Microbiology.
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    7. 7
      Yao, H.; Song, Y.; Chen, Y.; Wu, N.; Xu, J.; Sun, C.; Zhang, J.; Weng, T.; Zhang, Z.; Wu, Z.; Cheng, L.; Shi, D.; Lu, X.; Lei, J.; Crispin, M.; Shi, Y.; Li, L.; Li, S. Molecular architecture of the SARS-CoV-2 virus. Cell 2020, 183, 730– 738, e13  DOI: 10.1016/j.cell.2020.09.018
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      Molecular Architecture of the SARS-CoV-2 Virus
      Yao, Hangping; Song, Yutong; Chen, Yong; Wu, Nanping; Xu, Jialu; Sun, Chujie; Zhang, Jiaxing; Weng, Tianhao; Zhang, Zheyuan; Wu, Zhigang; Cheng, Linfang; Shi, Danrong; Lu, Xiangyun; Lei, Jianlin; Crispin, Max; Shi, Yigong; Li, Lanjuan; Li, Sai
      Cell (Cambridge, MA, United States) (2020), 183 (3), 730-738.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the mol. assembly of the authentic SARS-CoV-2 virus using cryoelectron tomog. (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were detd. to av. resolns. of 8.7-11 Å. Compns. of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diam. lumen.
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    8. 8
      Zhou, Y.; Jiang, S.; Du, L. Prospects for a MERS-CoV spike vaccine. Expert Rev. Vaccines 2018, 17, 677– 686,  DOI: 10.1080/14760584.2018.1506702
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      8
      Prospects for a MERS-CoV spike vaccine
      Zhou, Yusen; Jiang, Shibo; Du, Lanying
      Expert Review of Vaccines (2018), 17 (8), 677-686CODEN: ERVXAX; ISSN:1476-0584. (Taylor & Francis Ltd.)
      : Six years have passed since Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), a newly emerging infectious virus, was first reported in 2012. Although MERS-CoV has had a consistently high mortality rate in humans, no vaccines have been approved to prevent MERS-CoV infection in humans. MERS-CoV spike (S) protein is a key target for development of MERS vaccines.: In this review, we illustrate the structure and function of S protein as a vaccine target, describe available animal models for evaluating MERS vaccines, and summarize recent progress on MERS-CoV S-based vaccines, focusing on their ability to elicit antibody and/or cellular immune responses, neutralizing antibodies, and protection against MERS-CoV infection in different models. Prospects for future MERS-CoV S-based vaccines are discussed.: The majority of MERS vaccines under development are based on MERS-CoV S protein, including full-length S, S1, and receptor-binding domain (RBD). While it is essential to evaluate the safety of full-length S and S1-based MERS vaccines, further improvement of the efficacy of RBD-based vaccines using novel strategies would be necessary. Overall, this review provides informative guidance for designing and developing safe and effective MERS vaccines based on viral S protein.
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    9. 9
      Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; Wang, X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 2020, 581, 215– 220,  DOI: 10.1038/s41586-020-2180-5
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      9
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
      Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
      Nature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
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    10. 10
      Li, F.; Li, W.; Farzan, M.; Harrison, S. C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 2005, 309, 1864– 1868,  DOI: 10.1126/science.1116480
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      10
      Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor
      Li, Fang; Li, Wenhui; Farzan, Michael; Harrison, Stephen C.
      Science (Washington, DC, United States) (2005), 309 (5742), 1864-1868CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resoln. of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The at. details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
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    11. 11
      Liu, S.; Xiao, G.; Chen, Y.; He, Y.; Niu, J.; Escalante, C. R.; Xiong, H.; Farmar, J.; Debnath, A. K.; Tien, P.; Jiang, S. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 2004, 363, 938– 947,  DOI: 10.1016/s0140-6736(04)15788-7
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      11
      Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors
      Liu, Shuwen; Xiao, Gengfu; Chen, Yibang; He, Yuxian; Niu, Jinkui; Escalante, Carlos R.; Xiong, Huabao; Farmar, James; Debnath, Asim K.; Tien, Po; Jiang, Shibo
      Lancet (2004), 363 (9413), 938-947CODEN: LANCAO; ISSN:0140-6736. (Elsevier Science Ltd.)
      Studies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-assocd. coronavirus (SARS-CoV). We tested the inhibitory activity against infection of two sets of peptides corresponding to sequences of SARS-CoV spike protein HR1 and HR2 regions and investigated the interactions between the HR1 and HR2 peptides by surface plasmon resonance, sedimentation equilibration anal., CD, native polyacrylamide-gel electrophoresis, size exclusion high-performance liq. chromatog., and computer-aided homol. modeling and mol. docking anal. One peptide, CP-1, derived from the HR2 region, inhibited SARS-CoV infection in the micromolar range. CP-1 bound with high affinity to a peptide from the HR1 region, NP-1. CP-1 alone had low α-helicity and self-assocd. to form a trimer in phosphate buffer (pH 7.2). CP-1 and NP-1 mixed in equimolar concns. formed a six-helix bundle, similar to the fusogenic core structure of HIV-1 gp41. After binding to the target cell, the transmembrane spike protein might change conformation by assocn. between the HR1 and HR2 regions to form an oligomeric structure, leading to fusion between the viral and target-cell membranes. At the pre-fusion intermediate state, CP-1 could bind to the HR1 region and interfere with the conformational changes, resulting in inhibition of SARS-CoV fusion with the target cells. CP-1 might be modifiable to increase its anti-SARS-CoV activity and could be further developed as an antiviral agent for treatment or prophylaxis of SARS-CoV infection.
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    12. 12
      Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell. Mol. Immunol. 2020, 17, 613– 620,  DOI: 10.1038/s41423-020-0400-4
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      12
      Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine
      Tai, Wanbo; He, Lei; Zhang, Xiujuan; Pu, Jing; Voronin, Denis; Jiang, Shibo; Zhou, Yusen; Du, Lanying
      Cellular & Molecular Immunology (2020), 17 (6), 613-620CODEN: CMIEAO; ISSN:1672-7681. (Nature Research)
      The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.
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    13. 13
      Dai, L.; Gao, G. F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 2021, 21, 73– 82,  DOI: 10.1038/s41577-020-00480-0
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      13
      Viral targets for vaccines against COVID-19
      Dai, Lianpan; Gao, George F.
      Nature Reviews Immunology (2021), 21 (2), 73-82CODEN: NRIABX; ISSN:1474-1733. (Nature Research)
      A review of the progress of which viral elements are used in COVID-19 vaccine candidates, why they might act as good targets for the immune system and the implications for protective immunity. Vaccines are urgently needed to control the coronavirus disease 2019 (COVID-19) pandemic and to help the return to pre-pandemic normalcy. A great many vaccine candidates are being developed, several of which have completed late-stage clin. trials and are reporting pos. results.
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    14. 14
      Khan, S.; Tombuloglu, H.; Hassanein, S. E.; Rehman, S.; Bozkurt, A.; Cevik, E.; Abdel-Ghany, S.; Nabi, G.; Ali, A.; Sabit, H. Coronavirus diseases 2019: current biological situation and potential therapeutic perspective. Eur. J. Pharmacol. 2020, 886, 1733447,  DOI: 10.1016/j.ejphar.2020.173447
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      Tombuloglu, H.; Sabit, H.; Al-Suhaimi, E.; Al Jindan, R.; Alkharsah, K. R. Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2. PLoS One 2021, 16, e0250942  DOI: 10.1371/journal.pone.0250942
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      15
      Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2
      Tombuloglu, Huseyin; Sabit, Hussein; Al-Suhaimi, Ebtesam; Al Jindan, Reem; Alkharsah, Khaled R.
      PLoS One (2021), 16 (4), e0250942CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-neg. and false-pos. results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clin. SARS-CoV-2 pos. samples. The results are in good agreement with the CDC authorized Cepheid's Xpert Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.
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    16. 16
      Ravi, N.; Cortade, D. L.; Ng, E.; Wang, S. X. Diagnostics for SARS-CoV-2 detection: a comprehensive review of the FDA-EUA COVID-19 testing landscape. Biosens. Bioelectron. 2020, 165, 112454,  DOI: 10.1016/j.bios.2020.112454
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      16
      Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape
      Ravi, Neeraja; Cortade, Dana L.; Ng, Elaine; Wang, Shan X.
      Biosensors & Bioelectronics (2020), 165 (), 112454CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      A review. The rapidly spreading outbreak of COVID-19 disease is caused by the SARS-CoV-2 virus, first reported in Dec. 2019 in Wuhan, China. As of June 17, 2020, this virus has infected over 8.2 million people but ranges in symptom severity, making it difficult to assess its overall infection rate. There is a need for rapid and accurate diagnostics to better monitor and prevent the spread of COVID-19. In this review, we present and evaluate two main types of diagnostics with FDA-EUA status for COVID-19: nucleic acid testing for detection of SARS-CoV-2 RNA, and serol. assays for detection of SARS-CoV-2 specific IgG and IgM patient antibodies, along with the necessary sample prepn. for accurate diagnoses. In particular, we cover and compare tests such as the CDC 2019-nCoV RT-PCR Diagnostic Panel, Cellex's qSARS-CoV-2 IgG/IgM Rapid Test, and point-of-care tests such as Abbott's ID NOW COVID-19 Test. Antibody testing is esp. important in understanding the prevalence of the virus in the community and to identify those who have gained immunity. We conclude by highlighting the future of COVID-19 diagnostics, which include the need for quant. testing and the development of emerging biosensors as point-of-care tests.
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      Abbott Diagnostics Scarborough Inc. BinaxNOWTM COVID-19 Ag CARD [package insert, EUA]; Scarborough: MA, USA, 2020.
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      LumiraDx Group Limited. LumiraDxTM SARS-CoV-2 Ag Test [package insert, EUA]; Dumyat Business Park Alloa FK10 2PB: U.K., 2020.
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      Becton, Dickinson and Company. BD VeritorTM System for Rapid Detection of SARS-CoV-2 [package insert, EUA]; Sparks-Glencoe: MD, USA, 2020.
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      Quidel Corporation. Sofia Severe Acute Respiratory Syndrome Antigen FIA [package insert, EUA]: San Diego, CA, USA, 2020.
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      There is no corresponding record for this reference.
    21. 21
      Barlev-Gross, M.; Weiss, S.; Ben-Shmuel, A.; Sittner, A.; Eden, K.; Mazuz, N.; Glinert, I.; Bar-David, E.; Puni, R.; Amit, S.; Kriger, O.; Schuster, O.; Alcalay, R.; Makdasi, E.; Epstein, E.; Noy-Porat, T.; Rosenfeld, R.; Achdout, H.; Mazor, O.; Israely, T.; Levy, H.; Mechaly, A. Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens. Anal. Bioanal. Chem. 2021, 413, 3501– 3510,  DOI: 10.1007/s00216-021-03298-4
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      21
      Spike vs. nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens
      Barlev-Gross, Moria; Weiss, Shay; Ben-Shmuel, Amir; Sittner, Assa; Eden, Keren; Mazuz, Noam; Glinert, Itai; Bar-David, Elad; Puni, Reut; Amit, Sharon; Kriger, Or; Schuster, Ofir; Alcalay, Ron; Makdasi, Efi; Epstein, Eyal; Noy-Porat, Tal; Rosenfeld, Ronit; Achdout, Hagit; Mazor, Ohad; Israely, Tomer; Levy, Haim; Mechaly, Adva
      Analytical and Bioanalytical Chemistry (2021), 413 (13), 3501-3510CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many antigen detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false pos. results. We developed a novel alternative spike-based antigen assay, comprising 4 high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an inhouse nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR pos. and neg. nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clin. setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs. specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results.
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    22. 22
      Aoki, K.; Nagasawa, T.; Ishii, Y.; Yagi, S.; Okuma, S.; Kashiwagi, K.; Maeda, T.; Miyazaki, T.; Yoshizawa, S.; Tateda, K. Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs. J. Infect. Chemother. 2021, 27, 613– 616,  DOI: 10.1016/j.jiac.2020.11.021
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      22
      Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs
      Aoki, Kotaro; Nagasawa, Tatsuya; Ishii, Yoshikazu; Yagi, Shintaro; Okuma, Sadatsugu; Kashiwagi, Katsuhito; Maeda, Tadashi; Miyazaki, Taito; Yoshizawa, Sadako; Tateda, Kazuhiro
      Journal of Infection and Chemotherapy (2021), 27 (4), 613-616CODEN: JICHFN; ISSN:1341-321X. (Elsevier B.V.)
      Expansion of the testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important issue to mitigate the pandemic of coronavirus disease-2019 (COVID-19) caused by this virus. Recently, a sensitive quant. antigen test (SQT), Lumipulse SARS-CoV-2 Ag, was developed. It is a fully automated chemiluminescent enzyme immunoassay system for SARS-CoV-2. In this study, the anal. performance of SQT was examd. using clin. specimens from nasopharyngeal swabs using reverse transcription polymerase chain reaction (RT-PCR) as a control. Receiver operating characteristic anal. of 24 SARS-CoV-2-pos. and 524 -neg. patients showed an area under the curve of 0.957 ± 0.063. Using a cut-off value of 1.34 pg/mL, the sensitivity was 91.7%, the specificity was 98.5%, and the overall rate of agreement was 98.2%. In the distribution of neg. cases, the 99.5 percentile value was 1.03 pg/mL. There was a high correlation between the viral load calcd. using the cycle threshold value of RT-PCR and the concn. of antigen. The tendency for the antigen concn. to decrease with time after disease onset correlated with that of the viral load. Presented results indicate that SQT is highly concordant with RT-PCR and should be useful for the diagnosis of COVID-19 in any clin. setting. Therefore, this fully automated kit will contribute to the expansion of the testing capability for SARS-CoV-2.
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    23. 23
      Hirotsu, Y.; Maejima, M.; Shibusawa, M.; Nagakubo, Y.; Hosaka, K.; Amemiya, K.; Sueki, H.; Hayakawa, M.; Mochizuki, H.; Tsutsui, T.; Kakizaki, Y.; Miyashita, Y.; Yagi, S.; Kojima, S.; Omata, M. Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients. Int. J. Infect. Dis. 2020, 99, 397– 402,  DOI: 10.1016/j.ijid.2020.08.029
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      23
      Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients
      Hirotsu, Yosuke; Maejima, Makoto; Shibusawa, Masahiro; Nagakubo, Yuki; Hosaka, Kazuhiro; Amemiya, Kenji; Sueki, Hitomi; Hayakawa, Miyoko; Mochizuki, Hitoshi; Tsutsui, Toshiharu; Kakizaki, Yumiko; Miyashita, Yoshihiro; Yagi, Shintaro; Kojima, Satoshi; Omata, Masao
      International Journal of Infectious Diseases (2020), 99 (), 397-402CODEN: IJIDF3; ISSN:1201-9712. (Elsevier Ltd.)
      In routine clin. practice, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is detd. by reverse-transcription PCR (RT-PCR). In the current pandemic, a more rapid and high-throughput method is in growing demand. Here, the authors validated the performance of a new antigen test (LUMIPULSE) based on chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients and 231 individual samples from 4 infected patients and 215 uninfected individuals) were analyzed for SARS-CoV-2 with quant. RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. The authors detd. the cutoff value for antigen detection using receiver operating characteristic curve anal. and compared the performance of the antigen test with that of RT-qPCR. The authors also compared the viral loads and antigen levels in serial samples from seven infected patients. Using RT-qPCR as the ref., the antigen test exhibited 55.2% sensitivity and 99.6% specificity, with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, resp. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline, along with viral clearance. This gradual decline was in contrast with the abrupt pos.-to-neg. and neg.-to-pos. status changes obsd. with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients.
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    24. 24
      Pollock, N. R.; Savage, T. J.; Wardell, H.; Lee, R.; Mathew, A.; Stengelin, M.; Sigal, G. B. Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay. J. Clin. Microbiol. 2021, 59, e03077– 20,  DOI: 10.1128/JCM.03077-20
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      24
      Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay
      Pollock, Nira R.; Savage, Timothy J.; Wardell, Hanna; Lee, Rose A.; Mathew, Anu; Stengelin, Martin; Sigal, George B.
      Journal of Clinical Microbiology (2021), 59 (4), e03077CODEN: JCMIDW; ISSN:1098-660X. (American Society for Microbiology)
      Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (CT) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clin. nasopharyngeal (NP) samples is unknown. An ultrasensitive, quant. electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD S-PLEX SARS-CoV-2 N assay) was used to measure Ag in clin. NP samples from adults and children previously tested by PCR. The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/mL and a cutoff of 0.32 pg/mL. Ag concns. measured in clin. NP samples (collected in 3.0 mL of media) ranged from less than 160 fg/mL to 2.7μg/mL. Log-transformed Ag concns. correlated tightly with CT values. In 35 adult and 101 pediatric PCR-pos. samples, the sensitivities were 91% (95% confidence interval, 77 to 98%) and 79% (70 to 87%), resp. In samples with a CT of ≤35, the sensitivities were 100% (88 to 100%) and 96% (88 to 99%), resp. In 50 adult and 40 pediatric PCR-neg. specimens, the specificities were 100% (93 to 100%) and 98% (87 to 100%), resp. Nucleocapsid concns. in clin. NP samples span 8 orders of magnitude and correlate closely with RNA concns. (CT values). The S-PLEX Ag assay showed 96 to 100% sensitivity in samples from children and adults with CT values of ≤35, and a specificity of 98 to 100%. These results clarify Ag concn. distributions in clin. samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.
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    25. 25
      Pekosz, A.; Cooper, C. K.; Parvu, V.; Li, M.; Andrews, J. C.; Manabe, Y. C.; Kodsi, S.; Leitch, J.; Gary, D. S.; Roger-Dalbert, C. Antigen-based testing but not real-time polymerase chain reaction correlates with Severe Acute Respiratory Syndrome Coronavirus 2 viral culture. Clin. Infect. Dis. 2021, 73, e2861– e2866,  DOI: 10.1093/cid/ciaa1706
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      25
      Antigen-based testing but not real-time polymerase chain reaction correlates with severe acute respiratory syndrome Coronavirus 2 viral culture
      Pekosz, Andrew; Parvu, Valentin; Li, Maggie; Andrews, Jeffrey C.; Manabe, Yukari C.; Kodsi, Salma; Gary, Devin S.; Roger-Dalbert, Celine; Leitch, Jeffry; Cooper, Charles K.
      Clinical Infectious Diseases (2021), 73 (9), e2861-e2866CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)
      Individuals can test pos. for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by mol. assays following the resoln. of their clin. disease. Recent studies indicate that SARS-CoV-2 antigen-based tests are likely to be pos. early in the disease course, when there is an increased likelihood of high levels of infectious virus. Upper respiratory specimens from 251 participants with coronavirus disease 2019 symptoms (≤7 days from symptom onset) were prospectively collected and tested with a lateral flow antigen test and a real-time polymerase chain reaction (rt-PCR) assay for detection of SARS-CoV-2. Specimens from a subset of the study specimens were utilized to det. the presence of infectious virus in the VeroE6TMPRSS2 cell culture model. The antigen test demonstrated a higher pos. predictive value (90%) than rt-PCR (70%) when compared to culture-pos. results. The pos. percentage agreement for detection of infectious virus for the antigen test was similar to rt-PCR when compared to culture results. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture positivity represents a significant advancement in detg. the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. SARS-CoV-2 antigen testing can facilitate low-cost, scalable, and rapid time-to-result, while providing good risk detn. of those who are likely harboring infectious virus, compared to rt-PCR.
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    26. 26
      Scohy, A.; Anantharajah, A.; Bodéus, M.; Kabamba-Mukadi, B.; Verroken, A.; Rodriguez-Villalobos, H. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J. Clin. Virol. 2020, 129, 104455,  DOI: 10.1016/j.jcv.2020.104455
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      26
      Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis
      Scohy, Anais; Anantharajah, Ahalieyah; Bodeus, Monique; Kabamba-Mukadi, Benoit; Verroken, Alexia; Rodriguez-Villalobos, Hector
      Journal of Clinical Virology (2020), 129 (), 104455CODEN: JCVIFB; ISSN:1386-6532. (Elsevier B.V.)
      Ensuring accurate diagnosis is essential to limit the spread of SARS-CoV-2 and for the clin. management of COVID-19. Although real-time reverse transcription polymerase chain reaction (RT- qPCR) is the current recommended lab. method to diagnose SARS-CoV-2 acute infection, several factors such as requirement of special equipment and skilled staff limit the use of these time-consuming mol. techniques. Recently, several easy to perform rapid antigen detection tests were developed and recommended in some countries as the first line of diagnostic. The aim of this study was to evaluate the performances of the Coris COVID-19 Ag Respi-Strip test, a rapid immunochromatog. test for the detection of SARS-CoV-2 antigen, in comparison to RT-qPCR. 148 Nasopharyngeal swabs were tested. Amongst the 106 pos. RT-qPCR samples, 32 were detected by the rapid antigen test, given an overall sensitivity of 30.2%. All the samples detected pos. with the antigen rapid test were also pos. with RT-qPCR. Higher viral loads are assocd. with better antigen detection rates. Unfortunately, the overall poor sensitivity of the COVID-19 Ag Respi-Strip does not allow using it alone as the frontline testing for COVID-19 diagnosis.
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    27. 27
      Tang, Y. W.; Schmitz, J. E.; Persing, D. H.; Stratton, C. W. Laboratory diagnosis of COVID-19: current issues and challenges. J. Clin. Microbiol. 2020, 58, 10– 1128,  DOI: 10.1128/JCM.00512-20
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      There is no corresponding record for this reference.
    28. 28
      Che, X.-y.; Qiu, L.-w.; Pan, Y.-x.; Wen, K.; Hao, W.; Zhang, L.-y.; Wang, Y.-d.; Liao, Z.-y.; Hua, X.; Cheng, V. C. C.; Yuen, K.-y. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with Severe Acute Respiratory Syndrome. J. Clin. Microbiol. 2004, 42, 2629– 2635,  DOI: 10.1128/jcm.42.6.2629-2635.2004
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      28
      Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome
      Che, Xiao-yan; Qiu, Li-wen; Pan, Yu-xian; Wen, Kun; Hao, Wei; Zhang, Li-ya; Wang, Ya-di; Liao, Zhi-yong; Hua, Xu; Cheng, Vincent C. C.; Yuen, Kwok-yung
      Journal of Clinical Microbiology (2004), 42 (6), 2629-2635CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)
      A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-assocd. coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich ELISA for the detection of SARS-CoV. The assay employed a mixt. of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clin. probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a std. to establish a detection sensitivity of approximated 50 pg/mL. The linear range of detection in clin. specimens was from 100 pg/mL to 3.2 ng/mL. Using a panel of sera collected at different points in time, the amt. of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serol. confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quant. antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.
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    29. 29
      Diao, B.; Wen, K.; Chen, J.; Liu, Y.; Yuan, Z.; Han, C.; Chen, J.; Pan, Y.; Chen, L.; Dan, Y.; Wang, J.; Chen, Y.; Deng, G.; Zhou, H.; Wu, Y. Diagnosis of acute respiratory syndrome coronavirus 2 infection by detection of nucleocapsid protein. medRxiv 2020, 20032524,  DOI: 10.1101/2020.03.07.20032524
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    30. 30
      Grant, B. D.; Anderson, C. E.; Williford, J. R.; Alonzo, L. F.; Glukhova, V. A.; Boyle, D. S.; Weigl, B. H.; Nichols, K. P. SARS-CoV-2 coronavirus nucleocapsid antigen-detecting half-strip lateral flow assay toward the development of point of care tests using commercially available reagents. Anal. Chem. 2020, 92, 11305– 11309,  DOI: 10.1021/acs.analchem.0c01975
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      30
      SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents
      Grant, Benjamin D.; Anderson, Caitlin E.; Williford, John R.; Alonzo, Luis F.; Glukhova, Veronika A.; Boyle, David S.; Weigl, Bernhard H.; Nichols, Kevin P.
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (16), 11305-11309CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clin. sensitive, may fill many of the gaps in the current RT-PCR testing regime, esp. in low- and middle-income countries (LMICs). To date, many serol. LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clin. sensitivity than serol. assays. To date, only a very small no. of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using com. available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equiv. to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.
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    31. 31
      Ahmadivand, A.; Gerislioglu, B.; Ramezani, Z.; Kaushik, A.; Manickam, P.; Ghoreishi, S. A. Functionalized terahertz plasmonic metasensors: femtomolar-level detection of SARS-CoV-2 spike proteins. Biosens. Bioelectron. 2021, 177, 112971,  DOI: 10.1016/j.bios.2021.112971
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      31
      Functionalized terahertz plasmonic metasensors: Femtomolar-level detection of SARS-CoV-2 spike proteins
      Ahmadivand, Arash; Gerislioglu, Burak; Ramezani, Zeinab; Kaushik, Ajeet; Manickam, Pandiaraj; Ghoreishi, S. Amir
      Biosensors & Bioelectronics (2021), 177 (), 112971CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      Effective and efficient management of human betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 virus infection i.e., COVID-19 pandemic, required sensitive and selective sensors with short sample-to-result durations for performing desired diagnostics. In this direction, one appropriate alternative approach to detect SARS-CoV-2 virus protein at low level i.e., femtomolar (fM) is exploring plasmonic metasensor technol. for COVID-19 diagnostics, which offers exquisite opportunities in advanced healthcare programs, and modern clin. diagnostics. The intrinsic merits of plasmonic metasensors stem from their capability to squeeze electromagnetic fields, simultaneously in frequency, time, and space. However, the detection of low-mol. wt. biomols. at low densities is a typical drawback of conventional metasensors that has recently been addressed using toroidal metasurface technol. This research is focused on the fabrication of a miniaturized plasmonic immunosensor based on toroidal electrodynamics concept that can sustain robustly confined plasmonic modes with ultranarrow lineshapes in the terahertz (THz) frequencies. By exciting toroidal dipole mode using our quasi-infinite metasurface and a judiciously optimized protocol based on functionalized gold nanoparticles (AuNPs) conjugated with the specific monoclonal antibody specific to spike protein (S1) of SARS-CoV-2 virus onto the metasurface, the resonance shifts for diverse concns. of the spike protein are monitored. Possessing mol. wt. around ∼76 kDa allowed to detect the presence of SARS-CoV-2 virus protein with significantly low as limit of detection (LoD) was achieved as ∼4.2 fM. We envisage that outcomes of this research will pave the way toward the use of toroidal metasensors as practical technologies for rapid and precise screening of SARS-CoV-2 virus carriers, symptomatic or asymptomatic, and spike proteins in hospitals, clinics, labs., and site of infection.
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    32. 32
      Seo, G.; Lee, G.; Kim, M. J.; Baek, S.-H.; Choi, M.; Ku, K. B.; Lee, C.-S.; Jun, S.; Park, D.; Kim, H. G.; Kim, S.-J.; Lee, J.-O.; Kim, B. T.; Park, E. C.; Kim, S. I. Rapid detection of COVID-19 causative virus (SARS-CoV-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor. ACS Nano 2020, 14, 5135– 5142,  DOI: 10.1021/acsnano.0c02823
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      32
      Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor
      Seo, Giwan; Lee, Geonhee; Kim, Mi Jeong; Baek, Seung-Hwa; Choi, Minsuk; Ku, Keun Bon; Lee, Chang-Seop; Jun, Sangmi; Park, Daeui; Kim, Hong Gi; Kim, Seong-Jun; Lee, Jeong-O.; Kim, Bum Tae; Park, Edmond Changkyun; Kim, Seung Il
      ACS Nano (2020), 14 (4), 5135-5142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for contg. the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clin. samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was detd. using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concns. of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clin. transport medium. In addn., the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 x 101 pfu/mL) and clin. samples (LOD: 2.42 x 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunol. diagnostic method for COVID-19 that requires no sample pretreatment or labeling.
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    33. 33
      Zhong, J.; Rösch, E. L.; Viereck, T.; Schilling, M.; Ludwig, F. Toward rapid and sensitive detection of SARS-CoV-2 with functionalized magnetic nanoparticles. ACS Sens. 2021, 6, 976– 984,  DOI: 10.1021/acssensors.0c02160
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      33
      Toward Rapid and Sensitive Detection of SARS-CoV-2 with Functionalized Magnetic Nanoparticles
      Zhong, Jing; Roesch, Enja Laureen; Viereck, Thilo; Schilling, Meinhard; Ludwig, Frank
      ACS Sensors (2021), 6 (3), 976-984CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global medical systems and economies and rules our daily living life. Controlling the outbreak of SARS-CoV-2 has become 1 of the most important and urgent strategies throughout the whole world. As of Oct. 2020, there have not yet been any medicines or therapies to be effective against SARS-CoV-2. Thus, rapid and sensitive diagnostics is the most important measures to control the outbreak of SARS-CoV-2. Homogeneous biosensing based on magnetic nanoparticles (MNPs) is 1 of the most promising approaches for rapid and highly sensitive detection of biomols. This paper proposes an approach for rapid and sensitive detection of SARS-CoV-2 with functionalized MNPs via the measurement of their magnetic response in an ac magnetic field. For proof of concept, mimic SARS-CoV-2 consisting of spike proteins and polystyrene beads are used for expts. Exptl. results demonstrate that the proposed approach allows the rapid detection of mimic SARS-CoV-2 with a limit of detection of 0.084 nM (5.9 fmol). The proposed approach has great potential for designing a low-cost and point-of-care device for rapid and sensitive diagnostics of SARS-CoV-2.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitV2ku78%253D&md5=e79da90b2367b4017b15e4e00dae2277
    34. 34
      Ventura, B. D.; Cennamo, M.; Minopoli, A.; Campanile, R.; Censi, S. B.; Terracciano, D.; Portella, G.; Velotta, R. Colorimetric test for fast detection of SARS-CoV-2 in nasal and throat swabs. ACS Sens. 2020, 5, 3043– 3048,  DOI: 10.1021/acssensors.0c01742
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      34
      Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs
      Ventura, Bartolomeo Della; Cennamo, Michele; Minopoli, Antonio; Campanile, Raffaele; Censi, Sergio Bolletti; Terracciano, Daniela; Portella, Giuseppe; Velotta, Raffaele
      ACS Sensors (2020), 5 (10), 3043-3048CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biol. material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal soln. of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting 3 surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a soln. contg. the viral particle. The optical d. of the mixed soln. measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold std. for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiol., as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concn.
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    35. 35
      Pramanik, A.; Gao, Y.; Patibandla, S.; Mitra, D.; McCandless, M. G.; Fassero, L. A.; Gates, K.; Tandon, R.; Chandra Ray, P. The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles. Nanoscale Adv. 2021, 3, 1588– 1596,  DOI: 10.1039/d0na01007c
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      35
      The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles
      Pramanik, Avijit; Gao, Ye; Patibandla, Shamily; Mitra, Dipanwita; McCandless, Martin G.; Fassero, Lauren A.; Gates, Kalein; Tandon, Ritesh; Chandra Ray, Paresh
      Nanoscale Advances (2021), 3 (6), 1588-1596CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease that began in 2019 (COVID-19), has been responsible for 1.4 million deaths worldwide as of 13 Nov. 2020. Because at the time of writing no vaccine is yet available, a rapid diagnostic assay is very urgently needed. Herein, we present the development of anti-spike antibody attached gold nanoparticles for the rapid diagnosis of specific COVID-19 viral antigen or virus via a simple colorimetric change observation within a 5 min time period. For rapid and highly sensitive identification, surface enhanced Raman spectroscopy (SERS) was employed using 4-aminothiophenol as a reporter mol., which is attached to the gold nanoparticle via an Au-S bond. In the presence of COVID-19 antigen or virus particles, owing to the antigen-antibody interaction, the gold nanoparticles undergo aggregation, changing color from pink to blue, which allows for the detn. of the presence of antigen or virus very rapidly by the naked eye, even at concns. of 1 ng (ng) per mL for COVID-19 antigen and 1000 virus particles per mL for SARS-CoV-2 spike protein pseudotyped baculovirus. Importantly, the aggregated gold nanoparticles form "hot spots" to provide very strong SERS signal enhancement from anti-spike antibody and 4-aminothiophenol attached gold nanoparticles via light-matter interactions. Finite-difference time-domain (FDTD) simulation data indicate a 4-orders-of-magnitude Raman enhancement in "hot spot" positions when gold nanoparticles form aggregates. Using a portable Raman analyzer, our reported data demonstrate that our antibody and 4-aminothiophenol attached gold nanoparticle-based SERS probe has the capability to detect COVID-19 antigen even at a concn. of 4 pg (pg) per mL and virus at a concn. of 18 virus particles per mL within a 5 min time period. Using HEK293T cells, which express angiotensin-converting enzyme 2 (ACE2), by which SARS-CoV-2 enters human cells, we show that anti-spike antibody attached gold nanoparticles have the capability to inhibit infection by the virus. Our reported data show that antibody attached gold nanoparticles bind to SARS-CoV-2 spike protein, thereby inhibiting the virus from binding to cell receptors, which stops virus infection and spread. It also has the capability to destroy the lipid membrane of the virus.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVOgu7s%253D&md5=610835de6dba6d0c6c2a97d2daf2339f
    36. 36
      Yousefi, H.; Mahmud, A.; Chang, D.; Das, J.; Gomis, S.; Chen, J. B.; Wang, H.; Been, T.; Yip, L.; Coomes, E.; Li, Z.; Mubareka, S.; McGeer, A.; Christie, N.; Gray-Owen, S.; Cochrane, A.; Rini, J. M.; Sargent, E. H.; Kelley, S. O. Detection of SARS-CoV-2 viral particles using direct, reagent-free electrochemical sensing. J. Am. Chem. Soc. 2021, 143, 1722– 1727,  DOI: 10.1021/jacs.0c10810
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      36
      Detection of SARS-CoV-2 Viral Particles Using Direct, Reagent-Free Electrochemical Sensing
      Yousefi, Hanie; Mahmud, Alam; Chang, Dingran; Das, Jagotamoy; Gomis, Surath; Chen, Jenise B.; Wang, Hansen; Been, Terek; Yip, Lily; Coomes, Eric; Li, Zhijie; Mubareka, Samira; McGeer, Allison; Christie, Natasha; Gray-Owen, Scott; Cochrane, Alan; Rini, James M.; Sargent, Edward H.; Kelley, Shana O.
      Journal of the American Chemical Society (2021), 143 (4), 1722-1727CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combating the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold std. nucleic acid-based approaches require enzymic amplification to achieve clin. relevant levels of sensitivity and are not typically used outside of a lab. setting. We report reagent-free viral sensing that directly reads out the presence of viral particles in 5 min using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a neg. charged DNA linker that also features a tethered redox probe. When a pos. potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the 1st virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its assocd. spike protein in test samples and in unprocessed patient saliva.
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    37. 37
      Lee, J.-H.; Choi, M.; Jung, Y.; Lee, S. K.; Lee, C.-S.; Kim, J.; Kim, J.; Kim, N. H.; Kim, B.-T.; Kim, H. G. A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2). Biosens. Bioelectron. 2021, 171, 112715,  DOI: 10.1016/j.bios.2020.112715
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      37
      A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)
      Lee, Jong-Hwan; Choi, Minsuk; Jung, Yujin; Lee, Sung Kyun; Lee, Chang-Seop; Kim, Jung; Kim, Jongwoo; Kim, Nam Hoon; Kim, Bum-Tae; Kim, Hong Gi
      Biosensors & Bioelectronics (2021), 171 (), 112715CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a newly emerging human infectious disease. Because no specific antiviral drugs or vaccines are available to treat COVID-19, early diagnostics, isolation, and prevention are crucial for contg. the outbreak. Mol. diagnostics using reverse transcription polymerase chain reaction (RT-PCR) are the current gold std. for detection. However, viral RNAs are much less stable during transport and storage than proteins such as antigens and antibodies. Consequently, false-neg. RT-PCR results can occur due to inadequate collection of clin. specimens or poor handling of a specimen during testing. Although antigen immunoassays are stable diagnostics for detection of past infection, infection progress, and transmission dynamics, no matched antibody pair for immunoassay of SARS-CoV-2 antigens has yet been reported. In this study, we designed and developed a novel rapid detection method for SARS-CoV-2 spike 1 (S1) protein using the SARS-CoV-2 receptor ACE2, which can form matched pairs with com. available antibodies. ACE2 and S1-mAb were paired with each other for capture and detection in a lateral flow immunoassay (LFIA) that did not cross-react with SARS-CoV Spike 1 or MERS-CoV Spike 1 protein. The SARS-CoV-2 S1 (<5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86 x 105 copies/mL in the clin. specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects. This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFOisbzJ&md5=3f9071ac5e872a5880248ea47a10f3eb
    38. 38
      Pinals, R. L.; Ledesma, F.; Yang, D.; Navarro, N.; Jeong, S.; Pak, J. E.; Kuo, L.; Chuang, Y.-C.; Cheng, Y.-W.; Sun, H.-Y.; Landry, M. P. Rapid SARS-CoV-2 spike protein detection by carbon nanotube-based near-infrared nanosensors. Nano Lett. 2021, 21, 2272– 2280,  DOI: 10.1021/acs.nanolett.1c00118
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      Rapid SARS-CoV-2 spike protein detection by carbon nanotube-based near-infrared nanosensors
      Pinals, Rebecca L.; Ledesma, Francis; Yang, Darwin; Navarro, Nicole; Jeong, Sanghwa; Pak, John E.; Kuo, Lili; Chuang, Yung-Chun; Cheng, Yu-Wei; Sun, Hung-Yu; Landry, Markita P.
      Nano Letters (2021), 21 (5), 2272-2280CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      To effectively track and eliminate COVID-19, it is crit. to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXltFWjs78%253D&md5=d62b5397687debc14aac2e3e87583660
    39. 39
      Baker, A. N.; Richards, S.-J.; Guy, C. S.; Congdon, T. R.; Hasan, M.; Zwetsloot, A. J.; Gallo, A.; Lewandowski, J. R.; Stansfeld, P. J.; Straube, A.; Walker, M.; Chessa, S.; Pergolizzi, G.; Dedola, S.; Field, R. A.; Gibson, M. I. The SARS-COV-2 spike protein binds sialic acids and enables rapid detection in a lateral flow point of care diagnostic device. ACS Cent. Sci. 2020, 6, 2046– 2052,  DOI: 10.1021/acscentsci.0c00855
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      The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device
      Baker, Alexander N.; Richards, Sarah-Jane; Guy, Collette S.; Congdon, Thomas R.; Hasan, Muhammad; Zwetsloot, Alexander J.; Gallo, Angelo; Lewandowski, Jozef R.; Stansfeld, Phillip J.; Straube, Anne; Walker, Marc; Chessa, Simona; Pergolizzi, Giulia; Dedola, Simone; Field, Robert A.; Gibson, Matthew I.
      ACS Central Science (2020), 6 (11), 2046-2052CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N-acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform. The spike-protein from SARS-COV-2 is shown to bind sialic acids, which is exploited to assemble a lateral flow diagnostic tool, using glycans rather than antibodies, as the recognition unit.
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      Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505– 510,  DOI: 10.1126/science.2200121
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      Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase
      Tuerk, Craig; Gold, Larry
      Science (Washington, DC, United States) (1990), 249 (4968), 505-10CODEN: SCIEAS; ISSN:0036-8075.
      A novel method was developed for rapidly selecting preferred binding sequences from a population of random sequences. The method is designated systematic evolution of ligands by exponential enrichment (SELEX). High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calcd. pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding consts. of these two RNA's to T4 DNA polymerase are equiv. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high affinity ligands could conceivably be developed for any target mol.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXlt1OltLY%253D&md5=a40a28db9df4a5da4c1b1652a34aa715
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      Ellington, A. D.; Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818– 822,  DOI: 10.1038/346818a0
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      In vitro selection of RNA molecules that bind specific ligands
      Ellington, Andrew D.; Szostak, Jack W.
      Nature (London, United Kingdom) (1990), 346 (6287), 818-22CODEN: NATUAS; ISSN:0028-0836.
      Subpopulations of RNA mols. that bind specifically to a variety of org. dyes have been isolated from a population of random sequence RNA mols. Roughly one in 1010 random sequence RNA mols. folds in such a way as to create a specific binding site for small ligands.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXitVGgsw%253D%253D&md5=522ad1b4f0284ee88173b02035f9e9c5
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      Robertson, D. L.; Joyce, G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 1990, 344, 467– 468,  DOI: 10.1038/344467a0
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      Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA
      Robertson, Debra L.; Joyce, Gerald F.
      Nature (London, United Kingdom) (1990), 344 (6265), 467-8CODEN: NATUAS; ISSN:0028-0836.
      The discovery of RNA enzymes has, for the 1st time, provided a single mol. that has both genetic and catalytic properties. Techniques were previously devised for the mutation, selection, and amplification of catalytic RNA, all of which can be performed rapidly in vitro. This report describes how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution expts. to be carried out in response to artificially imposed selection constraints. The subject of the investigation was the Tetrahymena ribozyme, a self-splicing group I intron derived from the large rRNA precursor of Tetrahymena thermophila that catalyzes sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. Variant forms of the enzyme were selected that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected mol. represents the discovery of the 1st RNA enzyme known to cleave single-stranded DNA specifically.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitlWltLk%253D&md5=9f3ef5e76f028aa84277861930d98189
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      Nguyen, V.-T.; Seo, H. B.; Kim, B. C.; Kim, S. K.; Song, C.-S.; Gu, M. B. Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers. Biosens. Bioelectron. 2016, 86, 293– 300,  DOI: 10.1016/j.bios.2016.06.064
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      Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers
      Nguyen, Van-Thuan; Seo, Ho Bin; Kim, Byoung Chan; Kim, Sang Kyung; Song, Chang-Seon; Gu, Man Bock
      Biosensors & Bioelectronics (2016), 86 (), 293-300CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      In this research, we report highly sensitive and specific sandwich-type SPR-based biosensor for the detection H5Nx whole viruses. A few of aptamers, for the first time, were successfully screened and characterized for whole avian influenza (AI) viruses, H5Nx, by using Multi-GO-SELEX method. The affinities of the aptamers developed in this study were ranged from 8×104 to 1×104 EID50/mL, and the aptamers IF22, IF23 were found to be specific to H5N1 and H5N8, resp. In addn., some flexible aptamers IF20, IF15, and IF10 were found to bind to the H5N1 and H5N2, H5N1 and H5N8, or H5N1, H5N2, and H5N8, resp. Moreover, aptamers IF10 and IF22 were found to bind H5N1 virus simultaneously and confirmed to bind the different site of the same H5N1 whole virus. Therefore, this pair of aptamers, IF10 and IF22, were successfully applied to develop the sandwich-type SPR-based biosensor assay which is rapid, accurate for the detection of AI whole virus from H5N1-infected feces samples. The min. detectible concn. of H5N1 whole virus was found to be 200 EID50/mL with this sandwich-type detection using the aptamer pair obtained in this study. In addn., the sensitivity of this biosensor was successfully enhanced by using the signal amplification with the secondary aptamer conjugated with gold nanoparticles.
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      Park, J.-W.; Jin Lee, S.; Choi, E.-J.; Kim, J.; Song, J.-Y.; Bock Gu, M. An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening. Biosens. Bioelectron. 2014, 51, 324– 329,  DOI: 10.1016/j.bios.2013.07.052
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      An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening
      Park, Jee-Woong; Jin Lee, Su; Choi, Eun-Jin; Kim, Jaejo; Song, Jae-Young; Bock Gu, Man
      Biosensors & Bioelectronics (2014), 51 (), 324-329CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      In this study, we successfully developed a ssDNA aptamer pairs by using an advanced immobilization-free SELEX method with affinity-based selection and counter-screening process at every round. By implementing this method, two different aptamers specifically binding to bovine viral diarrhea virus type 1 (BVDV type 1) with high affinity were successfully screened. This aptamer pair was applied to ultrasensitive detection platform for BVDV type 1 in a sandwich manner. The ultrasensitive detection of BVDV type 1 using one of aptamers conjugated with gold nanoparticles was obtained in aptamer-aptamer sandwich type sensing format, with the limit of detection of 800 copies/mL, which is comparable to a real-time PCR method.
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      Seo, H. B.; Gu, M. B. Aptamer-based sandwich-type biosensors. J. Biol. Eng. 2017, 11, 11,  DOI: 10.1186/s13036-017-0054-7
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      Aptamer-based sandwich-type biosensors
      Seo, Ho Bin; Gu, Man Bock
      Journal of Biological Engineering (2017), 11 (), 11/1-11/7CODEN: JBEOBZ; ISSN:1754-1611. (BioMed Central Ltd.)
      Sandwich-type biosensor platforms have drawn lots of attentions due to its superior features, compared to other platforms, in terms of its stable and reproducible responses and easy enhancement in the detection sensitivity. The sandwich-type assays can be developed by utilizing a pair of receptors, which bind to the different sites of the same target. In this mini-review paper, the sandwich-type biosensors using either pairs of aptamers or aptamer-antibody pairs are reviewed in terms of its targets and platforms, the schematic designs, and their anal. performance.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslyrt7o%253D&md5=b10920617d0342cd0717648a30003830
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      Song, Y.; Song, J.; Wei, X.; Huang, M.; Sun, M.; Zhu, L.; Lin, B.; Shen, H.; Zhu, Z.; Yang, C. Discovery of aptamers targeting the receptor-binding domain of the SARS-CoV-2 spike glycoprotein. Anal. Chem. 2020, 92, 9895– 9900,  DOI: 10.1021/acs.analchem.0c01394
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      Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
      Song, Yanling; Song, Jia; Wei, Xinyu; Huang, Mengjiao; Sun, Miao; Zhu, Lin; Lin, Bingqian; Shen, Haicong; Zhu, Zhi; Yang, Chaoyong
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (14), 9895-9900CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, resp. Simulated interaction modeling, along with competitive expts., suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.
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      Stanborough, T.; Given, F. M.; Koch, B.; Sheen, C. R.; Stowers-Hull, A. B.; Waterland, M. R.; Crittenden, D. L. Optical detection of CoV-SARS-2 viral proteins to sub-picomolar concentrations. ACS Omega 2021, 6, 6404– 6413,  DOI: 10.1021/acsomega.1c00008
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      47
      Optical detection of CoV-SARS-2 viral proteins to sub-picomolar concentrations
      Stanborough, Tamsyn; Given, Fiona M.; Koch, Barbara; Sheen, Campbell R.; Stowers-Hull, Andre Buzas; Waterland, Mark R.; Crittenden, Deborah L.
      ACS Omega (2021), 6 (9), 6404-6413CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample vols. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiol. relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample vols., takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concns. Control expts. performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response obsd. for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or lab. expertise and equipment.
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    48. 48
      Pramanik, A.; Gao, Y.; Patibandla, S.; Mitra, D.; McCandless, M. G.; Fassero, L. A.; Gates, K.; Tandon, R.; Ray, P. C. Aptamer conjugated gold nanostar-based distance-dependent nanoparticle surface energy transfer spectroscopy for ultrasensitive detection and inactivation of corona virus. J. Phys. Chem. Lett. 2021, 12, 2166– 2171,  DOI: 10.1021/acs.jpclett.0c03570
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      48
      Aptamer conjugated gold nanostar-based distance-dependent nanoparticle surface energy transfer spectroscopy for ultrasensitive detection and inactivation of corona virus
      Pramanik, Avijit; Gao, Ye; Patibandla, Shamily; Mitra, Dipanwita; McCandless, Martin G.; Fassero, Lauren A.; Gates, Kalein; Tandon, Ritesh; Ray, Paresh Chandra
      Journal of Physical Chemistry Letters (2021), 12 (8), 2166-2171CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been detd. to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.
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    49. 49
      Schmitz, A.; Weber, A.; Bayin, M.; Breuers, S.; Fieberg, V.; Famulok, M.; Mayer, G. A SARS-CoV-2 spike binding DNA aptamer that inhibits pseudovirus infection by an RBD-independent mechanism. Angew. Chem., Int. Ed. 2021, 60, 2– 9,  DOI: 10.1101/2020.12.23.424171
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      Yang, G.; Li, Z.; Mohammed, I.; Zhao, L.; Wei, W.; Xiao, H.; Guo, W.; Zhao, Y.; Qu, F.; Huang, Y. Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1. Signal Transduction Targeted Ther. 2021, 6, 227,  DOI: 10.1038/s41392-021-00649-6
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      50
      Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1
      Yang, Ge; Li, Ziyue; Mohammed, Irfan; Zhao, Liping; Wei, Wei; Xiao, Haihua; Guo, Weisheng; Zhao, Yongxiang; Qu, Feng; Huang, Yuanyu
      Signal Transduction and Targeted Therapy (2021), 6 (1), 227CODEN: STTTCB; ISSN:2059-3635. (Nature Research)
      Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1.
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    51. 51
      Li, J.; Zhang, Z.; Gu, J.; Stacey, H. D.; Ang, J. C.; Capretta, A.; Filipe, C. D. M.; Mossman, K. L.; Balion, C.; Salena, B. J.; Yamamura, D.; Soleymani, L.; Miller, M. S.; Brennan, J. D.; Li, Y. Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library. Nucleic Acids Res. 2021, 49, 7267– 7279,  DOI: 10.1093/nar/gkab574
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      51
      Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library
      Li, Jiuxing; Zhang, Zijie; Gu, Jimmy; Stacey, Hannah D.; Ang, Jann C.; Capretta, Alfredo; Filipe, Carlos D. M.; Mossman, Karen L.; Balion, Cynthia; Salena, Bruno J.; Yamamura, Deborah; Soleymani, Leyla; Miller, Matthew S.; Brennan, John D.; Li, Yingfu
      Nucleic Acids Research (2021), 49 (13), 7267-7279CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      We performed in vitro selection expts. to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with resp. Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation expts. revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.
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    52. 52
      Gupta, A.; Anand, A.; Jain, N.; Goswami, S.; Anantharaj, A.; Patil, S.; Singh, R.; Kumar, A.; Shrivastava, T.; Bhatnagar, S.; Medigeshi, G. R.; Sharma, T. K.; DBT India Consortium for COVID-19 Research A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2. Mol. Ther.--Nucleic Acids 2021, 26, 321– 332,  DOI: 10.1016/j.omtn.2021.06.014
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      A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2
      Gupta, Ankit; Anand, Anjali; Jain, Neha; Goswami, Sandeep; Anantharaj, Anbalagan; Patil, Sharanabasava; Singh, Rahul; Kumar, Amit; Shrivastava, Tripti; Bhatnagar, Shinjini; Medigeshi, Guruprasad R.; Sharma, Tarun Kumar
      Molecular Therapy--Nucleic Acids (2021), 26 (), 321-332CODEN: MTAOC5; ISSN:2162-2531. (Elsevier Inc.)
      The recent SARS-CoV-2 outbreak has been declared a global health emergency. It will take years to vaccinate the whole population to protect them from this deadly virus, hence the management of SARS-CoV-2 largely depends on the widespread availability of an accurate diagnostic test. Toward addressing the unmet need of a reliable diagnostic test in the current work by utilizing the power of Systematic Evolution of Ligands by EXponential enrichment, a 44-mer G-quadruplex-forming DNA aptamer against spike trimer antigen of SARS-CoV-2 was identified. The lead aptamer candidate (S14) was characterized thoroughly for its binding, selectivity, affinity, structure, and batch-to-batch variability by utilizing various biochem., biophys., and in silico techniques. S14 has demonstrated a low nanomolar KD, confirming its tight binding to a spike antigen of SARS-CoV-2. S14 can detect as low as 2 nM of antigen. The clin. evaluation of S14 aptamer on nasopharyngeal swab specimens (n = 232) has displayed a highly discriminatory response between SARS-CoV-2 infected individuals from the non-infected one with a sensitivity and specificity of ∼91% and 98%, resp. Importantly, S14 aptamer-based test has evinced a comparable performance with that of RT-PCR-based assay. Altogether, this study established the utility of aptamer technol. for the detection of SARS-CoV-2.
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      Alves Ferreira-Bravo, I.; DeStefano, J. J. Xeno-nucleic Acid (XNA) 2’-fluoro-arabino nucleic acid (FANA) aptamers to the receptor-binding domain of SARS-CoV-2 S protein block ACE2 binding. Viruses 2021, 13, 1983,  DOI: 10.3390/v13101983
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      53
      Xeno-Nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding
      Alves Ferreira-Bravo, Irani; DeStefano, Jeffrey J.
      Viruses (2021), 13 (10), 1983CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ∼79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equil. dissocn. consts. (KD,app) of 10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, resp. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per wt. basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.
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      Behbahani, M.; Mohabatkar, H.; Hosseini, B. In silico design of quadruplex aptamers against the spike protein of SARS-CoV-2. Inf. Med. 2021, 26, 100757,  DOI: 10.1016/j.imu.2021.100757
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      In silico design of quadruplex aptamers against the spike protein of SARS-CoV-2
      Behbahani Mandana; Mohabatkar Hassan; Hosseini Barumand
      Informatics in medicine unlocked (2021), 26 (), 100757 ISSN:2352-9148.
      Nucleic acid aptamers are short sequences of nucleic acid ligands that bind to a specific target molecule. Aptamers are experimentally nominated using the well-designed SELEX (systematic evolution of ligands by exponential enrichment) method. Here, we designed a new method for diagnosis and blocking SARS-CoV-2 based on G-quadruplex aptamer. This aptamer was developed against the receptor-binding domain (RBD) region of the spike protein. In the current study, ten quadruplex DNA aptamers entitled AP1, AP2, AP3, AP4, AP5, AP6, AP7, AP8, AP9, and AP10 were designed in silico and had high HADDOCK scores. One quadruplex aptamer sequence (AP1) was selected based on the interaction with RBD of SARS-CoV-2. Results showed that AP1 aptamer could be used as an agent in the diagnosis and therapy of SARS-CoV-2, although more works are still needed.
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      Devi, A.; Chaitanya, N. S. N. Designing of peptide aptamer targeting the receptor-binding domain of spike protein of SARS-CoV-2: an in silico study. Mol. Diversity 2021, 1– 13,  DOI: 10.1007/s11030-020-10171-6
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      Artyushenko, P. V.; Mironov, V. A.; Mironov, V. A.; Morozov, D. I.; Shchugoreva, I. A.; Borbone, N.; Tomilin, F. N.; Kichkailo, A. S. Computational approach to design of aptamers to the receptor binding domain of SARS-CoV-2. Sib. Med. Rev. 2021, 2, 66– 67,  DOI: 10.20333/25000136-2021-2-66-67
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      Kowada, A. Greater public health impact of COVID-19 antigen detection tests. BMC Med. 2021, 19, 82,  DOI: 10.1186/s12916-021-01956-z
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      Greater public health impact of COVID-19 antigen detection tests
      Kowada, Akiko
      BMC Medicine (2021), 19 (1), 82CODEN: BMMECZ; ISSN:1741-7015. (BioMed Central Ltd.)
      Early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevents widespread transmission, manages outbreak control, improves poor prognosis, and reduces mortality of the Coronavirus disease 2019 (COVID-19). Ricks et al. report in BMC Medicine greater healthcare cost-saving and health outcomes of antigen detection rapid diagnostic tests compared to nucleic acid tests for symptomatic persons. The main outcomes in the study are the health system costs and health impacts (deaths averted and infectious days isolated). Antigen-detection rapid diagnostic test-led strategy is compared to a strategy based on nucleic acid tests and clin. judgment.
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      Pomplun, S.; Jbara, M.; Quartararo, A. J.; Zhang, G.; Brown, J. S.; Lee, Y.-C.; Ye, X.; Hanna, S.; Pentelute, B. L. De novo discovery of high-affinity peptide binders for the SARS-CoV-2 spike protein. ACS Cent. Sci. 2021, 7, 156– 163,  DOI: 10.1021/acscentsci.0c01309
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      De novo discovery of high-affinity peptide binders for the SARS-CoV-2 spike protein
      Pomplun, Sebastian; Jbara, Muhammad; Quartararo, Anthony J.; Zhang, Genwei; Brown, Joseph S.; Lee, Yen-Chun; Ye, Xiyun; Hanna, Stephanie; Pentelute, Bradley L.
      ACS Central Science (2021), 7 (1), 156-163CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The β-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissocn. consts. Kd = 80-970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concns. in a biol. matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics. Using affinity selection-mass spectrometry, we discovered synthetic peptides with nanomolar affinity for the SARS-CoV-2-spike-RBD and selectivity over human proteins.
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      Kaur, N.; Singh, R.; Dar, Z.; Bijarnia, R. K.; Dhingra, N.; Kaur, T. Genetic comparison among various coronavirus strains for the identification of potential vaccine targets of SARS-CoV2. Infect., Genet. Evol. 2021, 89, 104490,  DOI: 10.1016/j.meegid.2020.104490
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      Genetic comparison among various coronavirus strains for the identification of potential vaccine targets of SARS-CoV2
      Kaur, Navpreet; Singh, Rimaljot; Dar, Zahid; Bijarnia, Rakesh Kumar; Dhingra, Neelima; Kaur, Tanzeer
      Infection, Genetics and Evolution (2021), 89 (), 104490CODEN: IGENCN; ISSN:1567-1348. (Elsevier B.V.)
      A review. On-going pandemic pneumonia outbreak COVID-19 has raised an urgent public health issue worldwide impacting millions of people with a continuous increase in both morbidity and mortality. The causative agent of this disease is identified and named as SARS-CoV2 because of its genetic relatedness to SARS-CoV species that was responsible for the 2003 coronavirus outbreak. The immense spread of the disease in a very small period demands urgent development of therapeutic and prophylactic interventions for the treatment of SARS-CoV2 infected patients. A plethora of research is being conducted globally on this novel coronavirus strain to gain knowledge about its origin, evolutionary history, and phylogeny. This review is an effort to compare genetic similarities and diversifications among coronavirus strains, which can hint towards the susceptible antigen targets of SARS-CoV2 to come up with the potential therapeutic and prophylactic interventions for the prevention of this public threat.
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    60. 60
      Loos, C.; Atyeo, C.; Fischinger, S.; Burke, J.; Slein, M. D.; Streeck, H.; Lauffenburger, D.; Ryan, E. T.; Charles, R. C.; Alter, G. Evolution of early SARS-CoV-2 and cross-coronavirus immunity. mSphere 2020, 5, e00622– 20,  DOI: 10.1128/mSphere.00622-20
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      60
      Evolution of early SARS-CoV-2 and cross-coronavirus immunity
      Loos, Carolin; Atyeo, Caroline; Fischinger, Stephanie; Burke, John; Slein, Matthew D.; Streeck, Hendrik; Lauffenburger, Douglas; Ryan, Edward T.; Charles, Richelle C.; Alter, Galit
      mSphere (2020), 5 (5), e00622-20CODEN: MSPHCI; ISSN:2379-5042. (American Society for Microbiology)
      The novel coronavirus, SARS-coronavirus (CoV)-2 (SARS-CoV-2), has caused over 17 million infections in just a few months, with disease manifestations ranging from largely asymptomatic infection to critically severe disease. The remarkable spread and unpredictable disease outcomes continue to challenge management of this infection. Among the hypotheses to explain the heterogeneity of symptoms is the possibility that exposure to other coronaviruses (CoVs), or overall higher capability to develop immunity against respiratory pathogens, may influence the evolution of immunity to SARS-CoV-2. Thus, we profiled the immune response across multiple coronavirus receptor binding domains (RBDs), respiratory viruses, and SARS-CoV-2, to det. whether heterologous immunity to other CoV-RBDs or other infections influenced the evolution of the SARS-CoV-2 humoral immune response. Overall changes in subclass, isotype, and Fc-receptor binding were profiled broadly across a cohort of 43 individuals against different coronaviruses-RBDs of SARS-CoV-2 and the more common HKU1 and NL63 viruses. We found rapid functional evolution of responses to SARS-CoV-2 over time, along with broad but relatively more time-invariant responses to the more common CoVs. Moreover, there was little evidence of correlation between SARS-CoV-2 responses and HKU1, NL63, and respiratory infection (influenza and respiratory syncytial virus) responses. These findings suggest that common viral infections including common CoV immunity, targeting the receptor binding domain involved in viral infection, do not appear to influence the rapid functional evolution of SARS-CoV-2 immunity, and thus should not impact diagnostics or shape vaccine-induced immunity.
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      Li, F. Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 2016, 3, 237– 261,  DOI: 10.1146/annurev-virology-110615-042301
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      Structure, Function, and Evolution of Coronavirus Spike Proteins
      Li, Fang
      Annual Review of Virology (2016), 3 (), 237-261CODEN: ARVNBS; ISSN:2327-0578. (Annual Reviews)
      The coronavirus spike protein is a multifunctional mol. machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two crit. functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells.
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      Andersen, K. G.; Rambaut, A.; Lipkin, W. I.; Holmes, E. C.; Garry, R. F. The proximal origin of SARS-CoV-2. Nat. Med. 2020, 26, 450– 452,  DOI: 10.1038/s41591-020-0820-9
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      The proximal origin of SARS-CoV-2
      Andersen, Kristian G.; Rambaut, Andrew; Lipkin, W. Ian; Holmes, Edward C.; Garry, Robert F.
      Nature Medicine (New York, NY, United States) (2020), 26 (4), 450-452CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)
      There is no expanded citation for this reference.
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    63. 63
      Ou, X.; Liu, Y.; Lei, X.; Li, P.; Mi, D.; Ren, L.; Guo, L.; Guo, R.; Chen, T.; Hu, J.; Xiang, Z.; Mu, Z.; Chen, X.; Chen, J.; Hu, K.; Jin, Q.; Wang, J.; Qian, Z. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 2020, 11, 1620,  DOI: 10.1038/s41467-020-15562-9
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      63
      Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV
      Ou, Xiuyuan; Liu, Yan; Lei, Xiaobo; Li, Pei; Mi, Dan; Ren, Lili; Guo, Li; Guo, Ruixuan; Chen, Ting; Hu, Jiaxin; Xiang, Zichun; Mu, Zhixia; Chen, Xing; Chen, Jieyong; Hu, Keping; Jin, Qi; Wang, Jianwei; Qian, Zhaohui
      Nature Communications (2020), 11 (1), 1620CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
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      Idili, A.; Parolo, C.; Alvarez-Diduk, R.; Merkoçi, A. Rapid and efficient detection of the SARS-CoV-2 spike protein using an electrochemical aptamer-based sensor. ACS Sens. 2021, 6, 3093– 3101,  DOI: 10.1021/acssensors.1c01222
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      64
      Rapid and Efficient Detection of the SARS-CoV-2 Spike Protein Using an Electrochemical Aptamer-Based Sensor
      Idili, Andrea; Parolo, Claudio; Alvarez-Diduk, Ruslan; Merkoci, Arben
      ACS Sensors (2021), 6 (8), 3093-3101CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      The availability of sensors able to rapidly detect SARS-CoV-2 directly in biol. fluids in a single step would allow performing massive diagnostic testing to track in real time and contain the spread of COVID-19. Motivated by this, here, we developed an electrochem. aptamer-based (EAB) sensor able to achieve the rapid, reagentless, and quant. measurement of the SARS-CoV-2 spike (S) protein. First, we demonstrated the ability of the selected aptamer to undergo a binding-induced conformational change in the presence of its target using fluorescence spectroscopy. Then, we engineered the aptamer to work as a bioreceptor in the EAB platform and we demonstrated its sensitivity and specificity. Finally, to demonstrate the clin. potential of the sensor, we tested it directly in biol. fluids (serum and artificial saliva), achieving the rapid (minutes) and single-step detection of the S protein in its clin. range.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhslegsLvI&md5=eed8021b69f60f966c493363ef2aa479
    65. 65
      Zakashansky, J. A.; Imamura, A. H.; Salgado, D. F., 2nd; Romero Mercieca, H. C.; Aguas, R. F. L.; Lao, A. M.; Pariser, J.; Arroyo-Currás, N.; Khine, M. Detection of the SARS-CoV-2 spike protein in saliva with Shrinky-Dink electrodes. Anal. Methods 2021, 13, 874– 883,  DOI: 10.1039/d1ay00041a
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      65
      Detection of the SARS-CoV-2 spike protein in saliva with Shrinky-Dink electrodes
      Zakashansky, Julia A.; Imamura, Amanda H.; Salgado, Darwin F., II; Romero Mercieca, Heather C.; Aguas, Raphael F. L.; Lao, Angelou M.; Pariser, Joseph; Arroyo-Curras, Netzahualcoyotl; Khine, Michelle
      Analytical Methods (2021), 13 (7), 874-883CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
      A review. Using the children's toy, Shrinky-Dink, we present an aptamer-based electrochem. (E-AB) assay that recognizes the spike protein of SARS-CoV-2 in saliva for viral infection detection. The low-cost electrodes are implementable at population scale and demonstrate detection down to 1 ag mL-1 of the S1 subunit of the spike protein.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjvVCru7c%253D&md5=42942831b25fbc59924f0f80d848b087
    66. 66
      Cennamo, N.; Pasquardini, L.; Arcadio, F.; Lunelli, L.; Vanzetti, L.; Carafa, V.; Altucci, L.; Zeni, L. SARS-CoV-2 spike protein detection through a plasmonic D-shaped plastic optical fiber aptasensor. Talanta 2021, 233, 122532,  DOI: 10.1016/j.talanta.2021.122532
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      66
      SARS-CoV-2 spike protein detection through a plasmonic D-shaped plastic optical fiber aptasensor
      Cennamo, Nunzio; Pasquardini, Laura; Arcadio, Francesco; Lunelli, Lorenzo; Vanzetti, Lia; Carafa, Vincenzo; Altucci, Lucia; Zeni, Luigi
      Talanta (2021), 233 (), 122532CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A specific aptameric sequence has been immobilized on short polyethyleneglycol (PEG) interface on gold nano-film deposited on a D-shaped plastic optical fiber (POFs) probe, and the protein binding has been monitored exploiting the very sensitive surface plasmon resonance (SPR) phenomenon. The receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein has been specifically used to develop an aptasensor. Surface anal. techniques coupled to fluorescence microscopy and plasmonic anal. have been utilized to characterize the biointerface. Spanning a wide protein range (25 ÷ 1000 nM), the SARS-Cov-2 spike protein was detected with a Limit of Detection (LoD) of about 37 nM. Different interferents (BSA, AH1N1 hemagglutinin protein and MERS spike protein) have been tested confirming the specificity of our aptasensor. Finally, a preliminary test in dild. human serum encouraged its application in a point-of-care device, since POF-based aptasensor represent a potentially low-cost compact biosensor, characterized by a rapid response, a small size and could be an ideal lab. portable diagnostic tool.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFKrs77M&md5=918249e712869852a7e75668d1e1f903
    67. 67
      Aithal, S.; Mishriki, S.; Gupta, R.; Sahu, R. P.; Botos, G.; Tanvir, S.; Hanson, R. W.; Puri, I. K. SARS-CoV-2 detection with aptamer-functionalized gold nanoparticles. Talanta 2022, 236, 122841,  DOI: 10.1016/j.talanta.2021.122841
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      67
      SARS-CoV-2 detection with aptamer-functionalized gold nanoparticles
      Aithal, Srivatsa; Mishriki, Sarah; Gupta, Rohit; Sahu, Rakesh P.; Botos, George; Tanvir, Shazia; Hanson, Russell W.; Puri, Ishwar K.
      Talanta (2022), 236 (), 122841CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A rapid detection test for SARS-CoV-2 is urgently required to monitor virus spread and containment. Here, we describe a test that uses nanoprobes, which are gold nanoparticles functionalized with an aptamer specific to the spike membrane protein of SARS-CoV-2. An ELISA confirms aptamer binding with the spike protein on gold surfaces. Protein recognition occurs by adding a coagulant, where nanoprobes with no bound protein agglomerate while those with sufficient bound protein do not. Using plasmon absorbance spectra, the nanoprobes detect 16 nM and higher concns. of spike protein in phosphate-buffered saline. The time-varying light absorbance is examd. at 540 nm to det. the crit. coagulant concn. required to agglomerates the nanoprobes, which depends on the protein concn. This approach detects 3540 genome copies/μl of inactivated SARS-CoV-2.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFyjsbbP&md5=5f4e1f429d578a71f079547f697cb496

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c05521.

    • Recombinant proteins and aptamers used in this work; biosensors and assays developed for the detection of the SARS-CoV-2 S antigen; binding studies by direct ELAA to monitor S proteins interactions with aptamers; checkerboard titration of concentrations of capture and detection aptamers to optimize aptamer sandwich assay; sensitivity of the sandwich aptamer assay at different concentrations of reporter aptamer; and SARS-CoV-2 RNA detection in NP swab samples by droplet digital PCR analysis (PDF)


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