key: cord-0917006-8owxs9ut
authors: Zhang, Zijie; Pandey, Richa; Li, Jiuxing; Gu, Jimmy; White, Dawn; Stacey, Hannah D.; Ang, Jann C.; Steinberg, Catherine‐Jean; Capretta, Alfredo; Filipe, Carlos D. M.; Mossman, Karen; Balion, Cynthia; Miller, Matthew S.; Salena, Bruno J.; Yamamura, Deborah; Soleymani, Leyla; Brennan, John D.; Li, Yingfu
title: High‐Affinity Dimeric Aptamers Enable the Rapid Electrochemical Detection of Wild‐Type and B.1.1.7 SARS‐CoV‐2 in Unprocessed Saliva
date: 2021-10-04
journal: Angew Chem Int Ed Engl
DOI: 10.1002/anie.202110819
sha: ce5c58c5b156056bb71de7cc3820accfc0441778
doc_id: 917006
cord_uid: 8owxs9ut

We report a simple and rapid saliva‐based SARS‐CoV‐2 antigen test that utilizes a newly developed dimeric DNA aptamer, denoted as DSA1N5, that specifically recognizes the spike proteins of the wildtype virus and its Alpha and Delta variants with dissociation constants of 120, 290 and 480 pM, respectively, and binds pseudotyped lentiviruses expressing the wildtype and alpha trimeric spike proteins with affinity constants of 2.1 pM and 2.3 pM, respectively. To develop a highly sensitive test, DSA1N5 was immobilized onto gold electrodes to produce an electrochemical impedance sensor, which was capable of detecting 1000 viral particles per mL in 1:1 diluted saliva in under 10 min without any further sample processing. Evaluation of 36 positive and 37 negative patient saliva samples produced a clinical sensitivity of 80.5 % and specificity of 100 % and the sensor could detect the wildtype virus as well as the Alpha and Delta variants in the patient samples, which is the first reported rapid test that can detect any emerging variant of SARS‐CoV‐2.

As countries around the world strive to re-open their economies,t here is an eed to significantly enhance COVID-19 testing to both monitor and ideally prevent outbreaks and to allow contact tracing. Thec urrent testing regime in most countries is based on reverse transcription-polymerase chain reaction (RT-PCR) methods performed at centralized laboratories.S uch tests are sensitive but are costly,t oo complicated for operation by non-skilled laboratory technicians,and have long "sample-to-answer" times. [1] These tests are now being supplemented with rapid nucleic acid (e.g., Abbott ID NOW TM )a nd antigen tests (e.g., Abbott PanBio TM and Abbott BINAX NOW TM among others). However,these still require collection of nasal pharyngeal swabs (NPS) and have moderate performance metrics (sensitivity and specificity) relative to RT-PCR. [2] Therapid nucleic acid tests also require nucleic acid amplification, resulting in testing times of at least 30 minutes, [3] which makes them unsuitable for testing at points-of-entry,w orkplaces or congregate settings.

Existing rapid antigen tests provide fast sample-to-result times (5 to 13 min), [4] and because they detect the nucleocapsid protein, they can identify both the original SARS-CoV-2 virus as well as current variants of concern (VoCs) such as the B.1.1.7 (Alpha), B.1.351 (Beta) and P. 1( Gamma) variants. [5] However,while such tests show relatively good sensitivity and specificity for high viral loads, [6] they show poor performance when the viral load is low [7, 8] and have been shown to have very poor performance with saliva samples (sensitivity of 2-23 %), [9, 10] restricting them to NPS samples,and thus making it necessary to have samples collected by ah ealth-care professional. [11] Even using NPS samples,such tests are reported to be unsuitable for screening of asymptomatic patients and are not recommended for screening or surveillance in either home or congregate settings,such as schools or long-term care homes due to high false-negative rates. [9, 10] To enable rapid tests that can be performed either by the user or those without technical lab skills,several saliva-based test prototypes have been reported. There are several examples of both nucleic acid and antigen tests (typically nucleocapsid (N)-protein or spike (S)-protein), including lateral flow assays for genomic RNA [12] [13] [14] [15] [16] and S-protein, [17] as well as electrochemical sensors for detection of genomic RNA, [18] N-protein [19] or S-protein. [20] [21] [22] However,a tp resent these rapid saliva tests either require complicated and timeconsuming separation, [22] RNAe xtraction [15, 16] or amplification steps, [13, 18, 23] have not been validated with clinical samples, [21] require long assay times (> 1h), [12] [13] [14] [15] [16] or do not provide adequate detection limits (< 1000 viral copies per mL, [11, 17, [19] [20] [21] [22] [24] [25] [26] [27] [28] corresponding to aR T-PCR cycle threshold (C t )o f3 6). [29] Given these issues,t here remains an eed for simple,r apid and sensitive SARS-CoV-2 tests,w hich should be possible to achieve via detection of viral surface proteins (i.e., spike proteins) directly in unprocessed saliva.

Current saliva-based antigen tests targeting the S-protein typically show poor sensitivity,w hich in part is due to the affinity of monoclonal antibodies used as the capture and detection agents,w ith dissociation constants (K d )r anging from 1-10 nM. [30] [31] [32] To address this issue,our group and others have reported on the selection of anumber of DNAaptamers for the S1 subunit or receptor binding domain (RBD) of the SARS-CoV-2 spike protein. [33] [34] [35] [36] [37] [38] However,a ll aptamers reported to date show affinity similar to antibodies (K d ranges from 2-85 nM), and as such only afew aptamer-based rapid saliva tests for SARS-CoV-2 have been reported to date. [28, 39] We hypothesized that these issues could be solved from two approaches:( 1) deriving as pike-binding DNA aptamer with extremely high affinity as the molecular recognition element, and (2) utilizing an extremely sensitive signal transduction strategy to report the viral binding by the aptamer.T his rational approach was expected to overcome the loss of sensitivity reported for patient saliva samples, resulting in ar apid, simple and sensitive test for detecting SARS-CoV-2 from human saliva without amplification.

To derive ah igh-affinity DNAa ptamer,w et ook advantage of aw ell-established strategy of engineering bivalent or multivalent aptamers through ligation of two or more monomers, [40] [41] [42] [43] [44] [45] [46] [47] given the fact that the full spike protein is ahomotrimeric protein and each SARS-CoV-2 virus expresses % 30 spikes on its surface. [48, 49] We hypothesized that generation of dimeric aptamers from the best members in the collection of the diverse monomeric S1 protein-binding aptamers developed recently by our groups [38] may provide an aptamer with sufficient affinity to achieve this goal.

To develop ah ighly sensitive rapid test that works with real saliva samples,w ec hose to use Electrochemical Impedance Spectroscopy (EIS) as it is one of the most sensitive signal transduction strategies available. [50, 51] While several methods exist to produce electrochemical signals using aptamers, [52] [53] [54] the EIS readout provides ar apid, single-step detection method with high detection sensitivity using simple handheld instrumentation [55, 56] that can be scaled up for widespread use,making it ideally suited for developing rapid COVID-19 tests for use at home or in congregate settings.

Herein, we report on ar apid electrochemical test for SARS-CoV-2 using adimeric aptamer for direct detection of the spike protein in unprocessed saliva. Ther apid test, denoted as the Cov-eChip,has been evaluated with the spike proteins of the original SARS-CoV-2 virus as well as its Alpha and Delta variants,a nd has been validated using al arge number (> 70) of clinical saliva samples,d emonstrating performance exceeding any currently reported rapid test.

Construction of Dimeric Aptamers and Binding to the Trimeric Spike Protein of Wild-type SARS-CoV-2

Anumber of DNAaptamers were selected against the S1 subunit of the spike protein of the wildtype SARS-CoV-2 virus,a sr eported in our previous work, [38] several of which had dissociation constants (K d )i nt he range of 1-10 nM. Given the fact that (1) the spike protein of SARS-CoV-2 is at rimeric protein made of 3S 1-S2 monomers,( 2) each viral particle of SARS-CoV-2 contains % 30 spike proteins, [48, 49] and (3) our aptamers were selected using the monomeric S1 subunit, we hypothesized that dimeric aptamers could display much higher affinity for both the full-length trimeric spike protein and the intact virus.

We chose two Monomeric Spike-protein binding Aptamers,denoted as MSA1 and MSA5, to build dimeric aptamers as they exhibited the highest affinity to the S1 protein.

Specifically we constructed two homodimeric aptamers and one heterodimeric aptamer using MSA1T and MSA5T,t he truncated minimal sequences of the two aptamers that retained full activity (sequences and proposed secondary structures are shown in Figure 1A ;the sequences of all DNA oligonucleotides are shown in the Table S1 ). [38] These dimeric aptamers were generated by linking the MSA1T and MSA5T with apolythymidine (polyT) linker and are named DSA1N1, DSA5N5 and DSA1N5 (DSA: Dimeric Spike binding DNA Aptamer;N:and;1:MSA1T;5:MSA5T).

We first examined the effect of the linkers containing 10, 15, 20, 30 and 40 thymidines on the binding affinity of DSA1N5 to the wild-type trimeric spike protein (Wuhan variant, denoted as WHTS) using dot-blot assays.The dimeric aptamers containing al inker of 30, 40, and 20 thymidines showed similar binding affinity (K d of 0.12, 0.14 and 0.17 nM, respectively,F igure S1), but those with 15-and 10-thymidine linkers had poorer affinity (K d of 0.58 and 12.6 nM, respectively). DSA1N5 with the 30-T linker had ab inding affinity for the trimeric spike protein that is % 99-and 28-fold higher than the monomeric aptamers MSA1T and MSA5T,r espectively (Table S2 ). Ther esults indicate that the dimeric aptamer approach can produce aptamers with significantly enhanced affinity for the trimeric spike protein, presumably via bivalent recognition. [40] [41] [42] [43] [44] [45] [46] [47] Figure 1B shows binding curves derived from dot-blot assays using the full trimeric spike proteins of SARS-CoV-2 (representative dot blot assay results are provided in Figure S2 ). In comparison to the substantial affinity enhancement observed for the heterodimeric aptamer DSA1N5 ( % 99-fold;T able S2), the two homodimeric aptamers showed much reduced affinity enhancements relative to their monomeric counterparts (Table S2). As controls we also tested the scrambled sequences of the dimeric and monomeric aptamers (named as DMC,M1C and M5C) and no binding was observed for any of these controls ( Figure 1B and S2).

It is interesting to note that the affinity of MSA1T for the trimeric spike protein decreased 4.2-fold relative to the S1 subunit (K d of 11.9 nM vs.2 .8 nM;F igure S3A), but MSA5T had 2.9-fold higher affinity for the trimeric protein (K d of 3.4 nM vs.1 0.1 nM;F igure S3B), suggesting that the trimer formation affects the MSA1 binding epitope,and that MSA1 and MSA5 bind slightly different epitopes.T od etermine if MSA1T and MSA5T recognize the same epitope or different epitopes of the spike protein, we conducted ac ompetition assay that used non-radioactive MSA5T to compete with radioactive MSA1T ( Figure S4 ). We first incubated radioactive MSA1T with S1 under the condition where MSA1T was fully bound to S1, followed by the addition of increasing concentrations of MSA5T.T he results provided in Figure S4 clearly indicate that MSA5T can successfully compete with MSA1T,s uggesting that they recognize the same epitope of the S1 subunit, though, as noted above,i ti sl ikely that the exact epitopes may differ slightly.

In addition to the spike protein from the wild-type virus, we also tested the binding of the dimeric aptamer DSA1N5 with the spike protein of the B.1.1.7 Alpha variant that first emerged in the UK. DSA1N5 retained high affinity to the Alpha variant spike protein (denoted as UKTS, K d = 0.29 nM, Figure 1B ), suggesting that the 501Y mutation did not alter the binding epitope(s) of the dimeric aptamer. [57] Another current variant of concern is B.1.617.2 Delta variant, (also known as the Indian variant), which was first detected in India but now is in circulation around the world. [58] We investigated the binding of DSA1N5 with the spike protein of this variant and found that the dimeric aptamer still exhibited high affinity for the spike protein of this variant (denoted as INTS, K d = 0.48 nM, Figure 1B ). Because the S1 subunit of this variant was also available,w ec arried out comparison studies to examine the binding profiles of MSA1, MSA5 and DSA1N5 for binding to the S1 subunit of the wildtype SARS-CoV-2 (named WH-S1) and the Indian variant (named IN-S1). Thed ata provided in Figure S5 indicates that the two monomeric aptamers and the dimeric aptamer exhibited very similar binding affinities for WH-S1 and IN-S1. These results suggest that the dimeric aptamer can detect this hyper-transmissible and more immune-evasive variant. [59] Dimeric Aptamer Binding with Pseudotyped Lentiviruses of SARS-CoV-2

Each viral particle of SARS-CoV-2 carries multiple trimeric spike proteins and the average spacing distance of adjacent spike proteins on coronaviruses has been reported to be 13-15 nm. [60, 61] Thep redicted length of the dimeric aptamer DSA1N5 is greater than 15 nm. Therefore,i tw as expected that this aptamer would show enhanced affinity for viral particles.

To test for viral binding,weused apseudotyped lentivirus that was engineered to express the full trimeric S-protein of SARS-CoV-2. [62] [63] [64] Because the surface of this virus resembles that of SARS-CoV-2 and it can enter human cells but cannot replicate itself,i th as been used as as ubstitute for SARS-CoV-2. [62] Thes ame lentivirus that lacks the S-protein was used as acontrol virus for this experiment.

Using dot blot assays,w ee valuated DSA1N5 for viral recognition ( Figure 2 ). Thea nalysis of binding affinity showed that DSA1N5 had high affinity binding to the pseudotyped viruses carrying the wild-type Wuhan variant spike protein (denoted as WHPV), with a K d of 2.1 pM, a57fold improvement relative to the K d for the trimeric spike protein ( Figure 2 ). Thea dditional affinity improvement was attributed to the bivalent binding of the same aptamer to two different spike proteins on the viral surface.A sc ontrols,n o binding was observed between WHPV and DMC (an inactive mutant dimeric aptamer) or between DSA1N5 with the control lentivirus that does not express the spike protein of SARS-CoV-2 (CV). 

To produce Cov-eChips,terminal thiol modified aptamer DSA1N5-SH was bound to ag old electrode,w hich was backfilled with thiol terminated PEG 6000 to minimize nonspecific adsorption on the electrode surface.T he data on the characterization of the electrode are shown in Figure S6 . Gold electrodes were first cleaned and evaluated for reproducibility using cyclic voltammetry ( Figure S6A ). Theoverlapping reduction and oxidation peaks demonstrate the reproducibility of the electrodes.F ollowing each step of derivatization, the electrodes were evaluated using EIS and analyzed using the equivalent circuit model shown in Figure S7A , with the addition of the aptamer and PEG both showing an increase in the charge transfer resistance (R ct )d ue to the presence of passivating materials blocking the surface diffusion of redox reagents in the measurement solution ( Figure S6B and Figure S7B) . Note that the increase in R ct validates that the functionalization occurred, and all Cov-eChips were validated by this method prior to use.E IS was further utilized to optimize the concentration of the aptamer ( Figure S6C ), demonstrating an optimal concentration of 2 mMf or the aptamer solution. Thedensity of the aptamer on the electrode surface was determined using chrono-coulometry (Figure S6D) , providing as urface density value of (1.3 AE 0.2) 10 14 aptamers per cm 2 .N ote that the overlap in the graphs used for measuring the aptamer surface density also indicates that the aptamer functionalization is very uniform.

TheC ov-eChip sensor functionalized with DSA1N5 on the working electrode was used to measure the change in charge transfer resistance (denoted as the change in resistance over the initial resistance (DR ct /R ct )) of the Cov-eChip at different concentrations of the spike protein WHTS.I nt his case,the functionalized Cov-eChip acts as an electrochemical transducer that translates the binding of the spike protein to the aptamer-modified electrode to an increase in electrochemical impedance ( Figure 3A ). Initial studies evaluated the incubation time required for optimal detection of WHTS ( Figure 3B) , indicating an incubation time of as little as 5min could produce ameasurable signal with minimal background. Longer incubation times produced higher signals but also increased the background, and thus a5min incubation time was selected.

We then tested the detection of WHTS in binding buffer, which resulted in an enhanced R ct as WHTS concentrations increased, indicating reduced access of the redox reagents to the working electrode surface ( Figure 3C ). Figure 3D shows the changes in DR ct /R ct versus the concentration of WHTS, UKTS and INTS.F or these three proteins,t he log-linear range was 4f Mto4.4 pM with alimit of detection (LOD) of 1f Mf or WHTS,2 .8 fM for UKTS and 3.6 fM for INTS ( Figure 3D ). Thes ensitivity of the device for the WHTS (0.93), measured as the signal change (DR ct /R ct )p er log of concentration, however,i sc onsiderably higher than for UKTS (0.50) and INTS (0.65). Even so,t he results clearly show that the electrochemical assay can detect WHTS,UKTS We also examined the ability of the Cov-eChip functionalized with monomeric aptamer MSA1T or MSA5T to detect WHTS ( Figure S8) . TheLOD values were found to be 462 fM and 52 fM respectively,w hich were much worse than the LOD of 1f Mf or the dimeric aptamer Cov-eChip.A dditionally,t he sensitivity for MSA1T-Cov-eChip and MSA5T-Cov-eChip is 0.11 and 0.17, respectively,c ompared to 0.94 observed for DSA1N5-Cov-eChip.T hese results clearly indicate that the dimeric aptamer provides far superior detection sensitivity relative to its composite monomeric aptamers.

Thep erformance of DSA1N5-Cov-eChip was further validated using EIS to test human saliva samples (with a1:1 dilution) spiked with various concentrations of WHPV (Nyquist plots and electrochemical parameters are shown in Figures S9, panel Aa nd B) . Al og-linear dynamic range was exhibited in the 10 3 cp mL À1 to 10 5 cp mL À1 range and an LOD of 1000 cp mL À1 was calculated based on 3s of the background error ( Figure 4A ), again demonstrating superior detection sensitivity relative to the dot-blot assay.W ealso assessed the ability of DSA1N5-Cov-eChip to detect UKPV ( Figure 4A ). Theresult indicates that DSA1N5-Cov-eChip was capable of recognizing this variant PV,but with areduced detection limit (5000 cp mL À1 ;aloss of over 5-fold), but still on par with the LOD of other rapid tests for the WH variant. [17, [19] [20] [21] 24, 25, 28, 65] TheLOD of DSA1N5-Cov-eChip for WHPV was 2-3 orders of magnitude better than the monomeric aptamer sensors MSA1T-Cov-eChip (10 6 cp mL À1 )a nd MSA5T-Cov-eChip (10 5 cp mL À1 ;F igure S10).

DSA1N5-Cov-eChip was also challenged with other proteins (IL-6, IgG,S treptavidin), ac ontrol lentivirus not expressing spike proteins,and abacterium (S. aureus)spiked in human saliva to study the non-specific interaction of the Cov-eChip with non-target samples ( Figure 4B ). Thes ignal measured in response to the WHPV (! 10 3 cp mL À1 )w as significantly higher than measured with potentially interfering species,d emonstrating the capability of the aptamer to bind with the SARS-CoV-2 spike protein with high selectivity.

Another cross-reactivity study was done using as eries of negative patient saliva samples that were collected as shown in Figure 4C .Inthis case,the saliva samples were first spiked with 10 4 cp mL À1 of pseudotyped virus and then heat inactivated by treating them at 65 8 8Cfor 30 min to ensure operator safety and diluted 1:1w ith binding buffer.A ss hown in Figure S11 , the heating step causes as mall decrease in the measured signal ( % 20 %) and an increase in measurement error but enables safer sample handling of clinical samples in the laboratory.W hile the negative saliva samples showed ar elatively variable initial charge transfer resistance (0.8 AE 0.3), all samples showed substantial increases in the signal after spiking, with an average increase in charge transfer resistance of 0.9 AE 0.4 observed following the spiking of the saliva with the pseudotyped virus ( Figure 4D ). These results aptamer functionalized chip incubated with 10 3 cp mL À1 pseudotyped virus spiked diluted pooled saliva, Interleukin-6-1000 pg mL À1 spiked diluted pooled saliva, Streptavidin-1000 pg mL À1 spiked diluted pooled saliva, S. aureus-10 5 CFU mL À1 spiked diluted pooled saliva, control lentivirus-10 5 cp mL À1 spiked diluted pooled saliva, pseudotyped virus (10 3 -10 4 cp mL À1 )spiked diluted pooled saliva. C) Method for collection of patient saliva, patient sample treatment was done by heating the collected sample at 65 8 8Cf or 30 min followed by diluting in binding buffer (1:1 v/v %). D) Charge transfer resistance for 25 COVID-19n egative saliva samples before and after spiking with 10 4 cp mL À1 Wuhan pseudotyped virus. The error bars represent the standard deviationf rom the mean obtained using three (n = 3) separate devices per sample. clearly show that, although the variability of patient samples impacts the measured background signals,itisfeasible to use DSA1N5-Cov-eChip assay for detecting low levels of virus even in heterogeneous clinical saliva specimens.

To assess the clinical sensitivity and specificity of the Cov-eChip saliva assay,w ee xamined ap anel of 73 patient saliva samples (Table S3 ;3 6p ositives including 3A lpha variants and 2Delta variants;37negatives according to the nasopharyngeal swab RT-PCR results,and in line with the sample size requirements for obtaining emergencyu se authorization [66] ) in ab linded study.T he test procedure included:( 1) sample dilution in binding buffer to achieve 50 %s aliva content;(2) sample incubation on DSA1N5-Cov-eChip (5 min);( 3) washing the chip with binding buffer (1 min);( 4) acquiring the EIS signal readout in redox buffer (2 min) using amobileoperated potentiostat the size of aU SB stick ( Figure 5A ). Hence,t he sample-to-readout time,i ncluding sample processing,i sunder 10 min.

Te sting of the patient saliva samples collected from patients diagnosed with SARS-CoV-2 (positive) and those without the infection (negative) on the Cov-eChip showed that the DR CT /R CT values were clearly different between these groups:0 .5-1.2 for the negative group and 0.5-3.8 for the positive group ( Figure 5B ;the DR CT /R CT values measured for the saliva sample from each of the 73 patients are presented in Figure 5C ). We determined the clinical performance charac-teristics of this assay using the Receiver Operator Characteristics (ROC) curve (See Figure S12 ). [67] Theoverall accuracy, or area under the curve (AUC) was 0.923 (CI, 0.860-0.985) with an optimum sensitivity of 80.5 %( true positive cases detected) at athreshold of 1.27 DR ct /R ct and acorresponding specificity of 100 %( no false positive cases detected). These diagnostic performance characteristics meet the FDAr equirements for sensitivity (! 80 %) and specificity (99 %) for home-based antigen tests [68, 69] and surpasses the performance of many other antigen based rapid tests for detecting SARS-CoV-2 in saliva (Table S4 ), none of which tested am inimum of 30 positive and negative samples as required by FDA regulations.F urthermore,t his is the first report of any rapid tests being able to detect patients infected with the Alpha and Delta variants as positive for SARS-CoV-2.

Thedetermination of all our COVID-19 positive samples was made based on nasopharyngeal swab (NPS) RT-PCR data, which tests for viral RNA, while our test is designed to detect the spike protein on the viral particles in saliva. Therefore,itmight be possible that even though the NPS test was positive,there were not sufficient viruses in the saliva to be detected by the Cov-eChip assay.Although saliva samples were collected amedian of five days post NPS collection, this was not associated with the false-negative results.A nother possibility is that there were unknown inhibitory factors in the saliva that prevented the recognition of the spike protein by the aptamer in those samples that were determined to be positive by NPS RT-PCR. To test the latter hypothesis,w e further examined six false-negative samples (16, 26, 54, 66, 62 and 159) by spiking the WHPV into these samples at ac oncentration of 10 6 cp mL À1 .A tt his concentration, the Cov-eChip assay should provide aconvincing positive signal. However,a st he data provided in Figure S13 show,n one of these spiked samples produced asignificantly enhanced signal over the unspiked samples.These results strongly suggest that there were indeed inhibitory factors in these samples that prevented the aptamer recognition. Since these saliva samples were donated by hospitalized COVID patients,there is agood possibility that these saliva samples may contain spikebinding neutralizing antibodies,w hich can prevent the interaction of the aptamer with the spike protein. [70, 71] Saliva is also ah ighly diverse and variable fluid containing many proteins with ap rincipal one being mucin, ah eavily glycosylated protein. [72] Functionally,m ucins provide lubrication and ab arrier in the oral cavity against bacteria, viruses and environmental contaminants.T hese virus traps could therefore be another mechanism of interference to hinder aptamer binding. [73] Conclusion Given the ongoing surges in COVID-19 cases coupled with increasing reports of more highly transmissible and infective variants of concern, the need for asimple and rapid saliva-based SARS-CoV-2 test is more critical than ever. Herein, we have addressed this need by developing an ew electrochemical sensor based on adimeric DNAaptamer that can selectively bind the spike protein of both the Wuhan, Indian Delta and UK Alpha variants of the virus with high affinity,a llowing for detection of the SARS-CoV-2 virus directly in patient saliva samples in under 10 min with ac linical sensitivity of 80.5 %a nd specificity of 100 %. To the best of our knowledge,this is the first study that integrates ahigh affinity dimeric DNAaptamer with an electrochemical test to produce ultrasensitive rapid antigen tests,t he first example of an aptamer-based antigen test for SARS-CoV-2 that is validated with al arge number of unprocessed, real clinical saliva samples,and the first antigen test for detection of key variants of concern of SARS-CoV-2 including the Alpha and Delta variants.T he aptamer-based electrochemical sensor outperforms all current commercial and published rapid tests for SARS-CoV-2, including those that utilize NPS samples.T he ability to detect both the original as well as Alpha and Delta variants of the virus in an easily obtained saliva sample and to run the test without the need for complicated sample processing steps should allow implementation of the test in avariety of settings,including congregate settings (schools,l ong-term care homes), airports,a renas,o r even as ah ome-based test run by the test subject. Another advantage of the rapid test is the ability to perform diagnostic tests over time for the same patient to follow disease progression and determine when the virus is cleared. [74] It is well known that the viral load peaks several days postinfection and can remain above the infectious threshold for up to am onth following infection, [75] hence such al ongitudinal assessment can be useful both for catching infections early when viral load is still low,a nd also ensuring that patients isolate for asufficient period so as to not prematurely re-enter the community.T he use of DNAa ptamers as recognition elements is expected to make it relatively easy to adapt the test for future variants of concern by selecting new aptamers against variant S1 proteins followed by evaluation of the highest affinity dimeric constructs,a llowing the test to be rapidly deployed to allow screening of new variants.

Cochrane Database Syst. Rev. 2021, 3,C D013705

Angew.C hem. Int

COVID-19 antigen testing devices:N otice on minimum value for sensitivity

Policyf or Coronavirus Disease-2019T ests During the Public Health Emergency(Revised)

Emergency Use Authorization of Medical Products and Related Authorities

Human Salivary ProteomeW iki

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Theauthors declare no conflict of interest.Keywords: aptamers ·COVID-19 ·electrochemical biosensors · rapid tests ·s aliva