This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. pubs.acs.org/acssensors Article Rapid and Efficient Detection of the SARS-CoV‑2 Spike Protein Using an Electrochemical Aptamer-Based Sensor Andrea Idili, Claudio Parolo, Ruslán Alvarez-Diduk, and Arben Merkoçi* Cite This: https://doi.org/10.1021/acssensors.1c01222 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The availability of sensors able to rapidly detect SARS-CoV-2 directly in biological 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 electrochemical aptamer-based (EAB) sensor able to achieve the rapid, reagentless, and quantitative 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 clinical potential of the sensor, we tested it directly in biological fluids (serum and artificial saliva), achieving the rapid (minutes) and single-step detection of the S protein in its clinical range. KEYWORDS: aptasensors, electrochemical sensors, infectious diseases, EAB sensors, COVID-19 T he current COVID-19 pandemic has made it clear how a highly infectious airborne pathogen (such as SARS-CoV- 2) has the ability to spread globally in a matter of weeks.1,2 As used to diagnose COVID-19 is the polymerase chain reaction (PCR), which provides high sensitivity and specificity through the direct quantification of the viral RNA. This is a crucial influenza viruses, SARS-CoV-2 travels from patient to patient clinical parameter to estimate the stage of the infection and to within respiratory droplets (e.g., small aqueous particles of discover asymptomatic patients (i.e., people infected with saliva or mucus produced by exhalation), making it extremely COVID-19 that cannot be easily identified due to the absence challenging to contain due to its close relationship with social of symptoms).14 Despite these clinical advantages, the PCR distance (Figure 1A).3,4 Once the virus gets inside the requires trained personnel, expensive equipment, delicate organism, its membrane proteins (i.e., spike (S) protein) reagents, and a relatively long procedure, which hamper its interact with the transmembrane ACE2 receptor infecting the use for frequent testing (multiple times per week)15 and in host’s cells. The binding between the two proteins triggers the low-resource settings.9,10,16 These disadvantages make PCR cellular fusion of the virus and subsequent release of its genetic too slow for the immediate identification of infected material into the cytosol.2,5,6 This, in turn, allows the virus to asymptomatic individuals, leading to delays in the application replicate inducing tissue inflammation and leading to shortness of containment measures allowing the virus to spread further.15 of breath, chest pain, loss of speech, and eventually death As an alternative to the PCR, lateral flow immunoassays (Figure 1A).2,7 Besides the dramatic effects that this virus can (LFIAs) provide a more rapid response at the point of care,17 have on single individuals, we have also seen how a pandemic but their lower sensitivity and specificity relegate them can affect the entire world population by limiting movements primarily for end-point serologic applications (i.e., the and having a huge impact on the global economy.8,9 So how detection of anti-SARS-CoV-2 antibodies weeks after getting can we deal with such a catastrophe? Unfortunately, like any infected).18 Even the most recent LFIAs for antigenic testing other disease, finding the vaccine and cure need their time (in have considerably lower analytical performance (compared to the case of vaccine following the accelerated protocol, it took the PCR), leading to delayed and qualitative diagnosis, which at least one year); instead, specific diagnostic devices are available in weeks, providing monitoring tools to limit the spread of the disease (e.g., by introducing lockdowns and Received: June 10, 2021 evaluate the efficiency of prophylactic actions).9−11 Accepted: July 26, 2021 Current molecular approaches to infectious disease diagnosis are not rapid and decentralized enough to keep up with the spread rate of a highly infectious pathogen in a globalized world.12,13 For example, the current gold standard technique © XXXX American Chemical Society https://doi.org/10.1021/acssensors.1c01222 A ACS Sens. XXXX, XXX, XXX−XXX ACS Sensors pubs.acs.org/acssensors Article on a gold electrode surface (Figure 1B). A variation in the target concentration induces a change in the aptamer conformation, which in turn changes the position of the redox reporter (here, we used Atto MB2, which is a methylene blue derivative) relative to the electrode surface, generating a quantitative electrochemical signal.28−32 The precise analytical response of the EAB sensor is coupled with their quick response time (from few seconds to 5 min) and extremely simple operation (single step), making them ideal diagnostic devices for COVID-19 monitoring through high-frequency testing. Motivated by this, here, we describe the development and characterization of a new EAB sensor against the SARS- CoV-2 S protein and its ability to recognize the target in undiluted samples (serum and artificial saliva) in its clinical range. ■ RESULTS AND DISCUSSION As the recognition element for our sensor, we selected two recently developed DNA aptamers, termed 1C and 4C, able to recognize the receptor-binding domain (RBD) of the SARS- CoV-2 spike (S) protein.33 We selected these aptamers because they display three key features for the development of a responsive EAB sensor. First, the variants have been Figure 1. Ongoing COVID-19 pandemic is a direct effect of the selected in a working buffer that mimics the physiological appearance and the rapid global spreading of severe acute respiratory conditions; therefore, they can support the measurement of the syndrome coronavirus 2 (SARS-CoV-2).19 (A, left) The main human- S protein in biological fluids.21 Second, the estimated to-human transmission route is driven by respiratory droplets produced by the infected person (here, represented as the red dissociation constants (KD) of the aptamers (5.8 ± 0.8 nM person).2 The inhalation of these droplets allows the transmission of for 1C and 19.9 ± 2.6 nM for 4C) are comparable to those of the virus to another individual (blue healthy person). More commercially available antibodies developed to bind the S specifically, (A, right) SARS-CoV-2 can travel deeply in the protein.34 Third, the binding interactions between the target respiratory tract until reaching the lungs. There, because the viral and the aptamers have been previously characterized through particles cannot be recognized by the immune system,20 they start to the molecular dynamics (MD) technique.33 More specifically, interact with the cells through their membrane spike (S) proteins, the variants can interact with the S protein through the which can be recognized by the angiotensin-converting enzyme 2 formation of a network of hydrogen bonds creating two (ACE2) receptor. This binding first triggers the virus entry into the consecutive binding interfaces in the case of the 1C variant, patient’s cell through the cleavage of the S1/S2 site by surface and only one for the 4C variant.33 These data suggest that the transmembrane protease serine 2,2 and the next activation of endolysosomal cathepsin L to induce virus−cell membrane fusion. magnitude of the binding-induced conformational change in After this event, the RNA genome is released into the cytosol of the the 1C variant is stronger than that in the 4C variant. Although patient’s cell, where it is translated into the proteins, which can this promising structural prediction confirms the binding, it is activate the replication of the virus inside the patient’s cells. (B) not clear if these interactions can induce a conformational Electrochemical aptamer-based (EAB) sensors exploit the binding- change in the aptamers. Indeed, this structure-switching induced conformational change of a covalently attached, redox property is crucial to generate a detectable output signal on reporter-modified aptamer to generate an easily measurable electro- the EAB sensing platform (Figure 1B).28,35 chemical signal. Specifically, the changes in conformation alter the We used fluorescence spectroscopy to study if the rate with which the redox reporter (here, we used Atto MB2, a interaction with the target induces a conformational change derivative of methylene blue) exchange electrons with the inter- in the selected aptamers. Because the binding event should rogating electrode. Because this sensing mechanism is based on a binding-induced conformational change of the receptor, the platform bring their 5′-ends far away from the 3′-ends (with respect to results in reagentless, rapid, and selective measurements directly in their folded stem-loop state), we modified these termini with a undiluted biological fluids.21 pair of a fluorophore (6-FAM) and a quencher (BHQ-1) to track this structural variation (Figure 2A).36,37 As targets, we have used the RBD and the S protein for two reasons: (1) both in turn affects the way patients are managed.18 Therefore, the proteins share the same target protein domain recognized by development of biosensing platforms able to detect the the aptamers and (2) the detection of the larger S protein clinically relevant concentrations of SARS-CoV-2 in a single could demonstrate the ability of the aptamers to support the step directly in untreated biological fluids could represent a recognition of the virus. To characterize them, we monitored new clinical tool to achieve rapidly and efficiently contact the fluorescence signal of the aptamers in the presence of tracking of the COVID-19 outbreak.15 increasing concentrations of the targets (Figure S1). We found Recently, we and other researchers have been working on that the presence of the S protein induces an increase in the the development of a new type of sensing technology that can fluorescence signal, supporting the hypothesis that formation provide a precise quantitative response at the point of care: of the target−aptamer complex brings the fluorophore far away electrochemical aptamer-based (EAB) sensors.22−27 This from the quencher. On the contrary, for the smaller RBD technology relies on the signal produced by a binding-induced protein, we observe only a small suppression of the signal conformational change of a redox reporter-modified aptamer (Figure S1). We believe that this difference depends on the B https://doi.org/10.1021/acssensors.1c01222 ACS Sens. XXXX, XXX, XXX−XXX ACS Sensors pubs.acs.org/acssensors Article and 11 ± 5 nM for the 4C variant and both aptamers display a comparable signal change. However, we observe that the estimated affinities have slightly higher values than what was previously reported.33 We suspect that this could be due to the presence of the fluorophore−quencher pair in their sequences. Specifically, previous studies have demonstrated how the formation of the fluorophore−quencher complex could stabilize the native structure of DNA-based structures inducing a more folded conformation.40−42 To better understand the different binding behaviors observed with respect to the previous study, we have reduced the stability of the native conformation of the aptamers used for the fluorescent studies. Specifically, by reducing the electrostatic interactions between mono- and divalent cations (present in the buffer solution) with the negatively charged phosphate backbone, we balanced the stabilization produced by the fluorophore/quencher pair.40,41 To achieve this, we have adopted a new working buffer with a lower salt content but with the same amount of magnesium ions (2 mM, see the Materials and Methods section for the details). We decided to adopt this strategy to avoid modification of the aptamer sequences, which could negatively affect the variants’ binding activities. Then, to demonstrate the reduced stability of the aptamers we used thermal melting curve experiments. This apporach allows us to associate the aptamer stability with respect to its melting temperature (TM) value.43 Basically, we observed at which temperature and buffer solution 50% of the aptamer population unfolds due to the thermal denaturation process. This induces the fluorophore and the quencher to move apart leading to an increase in the fluorescence signal. Therefore, a lower or higher TM value can be associated with Figure 2. (A) We characterized the binding activities of aptamers the lower or higher thermodynamic stability of the aptamer against the SARS-CoV-2 RBD (black curves) and the S protein (red variants. As expected, we found both variants display lower curves) in solution using fluorescence spectroscopy. To do this, we labeled the 1C and 4C variants with a fluorophore−quencher couple melting temperatures when they are tested in the new buffer at the two ends. In the absence of the target, the close proximity supporting the validity of our approach (Figure S2). between the fluorophore and the quencher decreases the overall Next, we tested again the bioreceptors in the presence of fluorescent signal. Instead, the presence of the target induces a target proteins (Figures 2B and S3). We observe for both binding-induced conformational change in the aptamer structure, variants, a higher positive fluorescence signal change and lower separating the fluorophore from the quencher, thus increasing the KDs. Specifically, the 1C variant displays maximum signal gain fluorescent signal. (B) We find that the 1C aptamer exhibits a lower values of +40.3 ± 1.5 and +33.9 ± 3.8% and KD values of 4.8 ± dissociation constant (4.8 ± 0.8 nM) and a higher signal gain (+40.3 0.8 and 116 ± 34 nM for the S protein and the RBD (Figures ± 1.5%) for the S protein (red curves) than the C4 variant (KD = 25.4 2B, top and S3), respectively, while the 4C variant displays ± 10.5 nM; 13.6 ± 1.5%). As expected, the optically labeled variants signal gain values of +13.6 ± 1.5 and +13.1 ± 5.0% and KD can also recognize also the RBD target (black curves) but they are displaying lower affinities and signal gain due to the smaller size of the values of 25.4 ± 10.5 and 24 ± 23 nM for the S protein and the target. The binding curves were obtained by adding increasing RBD (Figures 2B, bottom and S3), respectively. The observed concentrations of protein targets to a 5 nM concentration of 1C (B, signal change further confirms the ability of the aptamers to top) or 4C variant (B, bottom) in 0.1× PBS buffer (NaCl 13.7 mM, undergo a binding-induced conformational change. Finally, KCl 0.27 mM, Na2HPO4 1 mM, and KH2PO4 0.18 mM), 2 mM since the 1C aptamer displays the higher signal gain and the MgCl2, pH 7.4 at 25 °C. lower KD for the S protein, we selected this variant for its next adaptation as a bioreceptor for the EAB sensing platform. To different sizes of the proteins and their ability to interact with this purpose, we replaced the fluorophore and the quencher their targets (e.g., ACE2 receptor) mainly through electrostatic with a thiol group at 5′-ends and Atto MB2 (a methylene blue and van der Waals interactions.38,39 Specifically, the S protein derivative) at 3′-ends as a redox tag (Figure S4). (78 kDa vs 23 kDa of the RBD) can interact with the The newly fabricated EAB sensor responds to the presence negatively charged aptamer through a higher binding interface, of the target protein when interrogated using square wave which could generate a different microenvironment, for voltammetry (SWV, Figure S5). To demonstrate the ability of example, in the presence of extra charged amino acids located the aptamer to undergo a binding-induced conformational in the proximity of the bound oligonucleotide. This could change upon its attachment on the gold electrode, we induce a stronger conformational change in the aptamer characterized the relationship between its signal change and structure. Fitting the collected data using a Langmuir isotherm the frequency of the interrogating SWV potential pulse.44 equation (see the Materials and Methods section), we can Previous studies25,26,45−47 clearly demonstrate that the SWV estimate with precision the KD associated with the S protein. frequency used during the sampling of the electrochemical Specifically, we find KD values of 26 ± 8 nM for the 1C variant signal is crucial for the optimization of EAB sensors. C https://doi.org/10.1021/acssensors.1c01222 ACS Sens. XXXX, XXX, XXX−XXX ACS Sensors pubs.acs.org/acssensors Article Specifically, this parameter affects the sign of the signal change larger response than the RBD. Specifically, the former target (i.e., generating either signal-off or signal-on behavior) and its displays maximum signal gain values of −5.2 ± 0.4 and of +7.3 magnitude (overall signal gain). This is mainly due to the ± 0.5% at 5 and 300 Hz, respectively, while the latter displays binding-induced changes in aptamer flexibility, which alters its signal gain values of −5.0 ± 0.2 and +5.1 ± 0.2% (Figure 2), electron transfer kinetic because the redox reporter could be respectively, for frequencies at 5 and 300 Hz. To understand pushed farther from or closer to the electrode surface upon the clinical value of the sensor, we estimated the associated binding with the target. Therefore, to explore this, we tested dissociation constants through the analysis of the binding our sensor in the presence of a saturating concentration of the curves (see the Materials and Methods section). We found that RBD (100 nM), and we collected its electrochemical response the aptamer displays different affinities or KDs for the two over square wave frequencies ranging from 5 to 1000 Hz targets. Specifically, the RBD results in KD values of 181 ± 43 (Figure S5). We find that the sensor’s signal displays a strong and 1231 ± 209 pM at 5 and 300 Hz, respectively, which is in dependency on the SWV frequency. Specifically, we observed agreement with the previous study (Figure 2).34,49 The larger S at a high frequency a signal-on response and at a low frequency protein displays a better sensitivity and KD values of 35.4 ± a signal-off response. This dual behavior further demonstrates 11.7 and 126.4 ± 50.4 pM at 300 and 5 Hz, respectively, which the presence of a binding-induced conformational change since cover the wide clinical relevant range.34,50 More specifically, it indicates the presence of two major aptamer conformation although the sensor is not able to achieve the same sensitivity populations. of molecular techniques based on enzymatic amplification such Our EAB sensors can recognize the S protein in its clinically as PCR or LAMP (from 1 to 100 copies/mL),51 its dynamic relevant range, providing a new analytical tool for the detection range cover a concentration range (from ≈760 pg/mL to 76 of SARS-CoV-2. To characterize our sensors, we recorded their ng/mL) that is comparable with point-of-care approaches SWV signal in the presence of increasing concentrations of the developed for the detection of SARS-CoV-2 and used for high- frequency testing.52,53 Moreover, we find that the estimated KD RBD and the S protein (Figure 3). We observe for both targets for the 1C variant on the surface is lower than what we have the expected Langmuir isotherm binding curves at all square obtained using fluorescence spectroscopy (Figure S3). We wave frequencies tested (5 and 300 Hz). Comparing the believe that this result could arise from two additive effects. collected data, we note that the S protein produces a slightly The first one is originated from the different labeling molecules used to modify the aptamer variant and track its conforma- tional change. As highlighted before, the fluorophore− quencher couple can stabilize the native conformation of the aptamer40−42 with respect to the redox molecule that does not affect its stability.54 The second effect arises from the ability of the larger S protein to strongly interact with surfaces through electrostatic and van der Waals interactions (i.e., not selected during the SELEX process). These combined effects lead to an improvement of the overall affinity of the variant on the electrode surface.55−57 Our EAB sensor displays enough specificity to recognize the S protein from different coronaviruses, indicating its potential use for diagnostic applications. We tested the selectivity of the sensor challenging it with two RBD proteins selected from previous coronaviruses (SARS-CoV and MERS-CoV) and the non-viral protein NGAL (a protein found in various bodily fluids27 including saliva, blood, and urine). We observe that the sensor at 300 Hz does not produce a detectable signal change for any of the three proteins over the range of concentrations tested (from 10 pM to 100 nM) (Figure 4A, top). On the contrary, at lower SWV frequencies (5 Hz), only the SARS- CoV-1 RBD protein induces a signal change of the sensor, Figure 3. Newly fabricated EAB sensor responds to the increasing concentrations of the SARS-CoV-2 RBD (black curves) and the S which is comparable to what is observed for SARS-CoV-2 protein (red curves) producing the expected Langmuir binding (Figure 4A, bottom). We believe that this result depends on curves. To support the electrochemical readout, we modified the the selected aptamer and the structural properties of the target. aptamer with an Atto MB2 redox reporter and attached it via a Specifically, the aptamer was selected without performing a hexanethiol anchor to a gold wire electrode coated with a self- counter-selection against the SARS-CoV RBD protein during assembled monolayer of 6-mercapto-1-hexanol. As previously the SELEX process,33 and at the same time the RBD target observed for sensors belonging to this class, our EAB sensor displays from SARS-CoV shares almost 70% of the overall structure a signal-on response at higher square wave frequencies (300 Hz) and with the SARS-CoV-2 RBD.58−61 Taking together these two a signal-off response at lower frequencies (5 Hz). At the same time, conditions, we believe that the SARS-CoV-1 RBD protein is the dynamic range (defined here as the range from 10 to 90% of the able to bind the aptamer receptor without inducing a maximum signal change) of the sensor allows covering the clinically relevant range of SARS-CoV-2 in infected individuals,48 demonstrat- conformational change; therefore, it can only decrease the ing its clinical value. The binding curves were obtained in PBS buffer, collisions between the variant and the electrode surface 2 mM MgCl2, pH 7.4 at 25 °C, using square wave voltammetry (through steric hindrance), leading in a signal-off behavior. (SWV). The error bars reflect standard deviations derived using at Despite this, the different signal responses at low and high least three independently fabricated sensors. SWV frequencies displayed by the sensor can be used to D https://doi.org/10.1021/acssensors.1c01222 ACS Sens. XXXX, XXX, XXX−XXX ACS Sensors pubs.acs.org/acssensors Article untreated biological fluids in vitro and in vivo.27,62,63 Harnessing this unique feature, we tested our sensor directly in artificial saliva and fetal bovine serum (FBS) (Figure 5). We Figure 4. Sensor is specific and rapid providing a new clinical tool for the detection of the SARS-CoV-2 S protein. (A) To demonstrate this, we challenged the EAB sensor with the viral protein SARS-CoV-1 RBD (green curves) and MERS RBD (black curves) and the protein biomarker NGAL (blue curves). At 300 Hz (A, top), we do not observe a detectable signal change from any interference proteins over the concentration range we tested. On the contrary, at 5 Hz, the SARS-COV RBD is the only protein able to induce a response in the sensor, which is similar to the SARS-CoV-2 S protein (red curves). We believe that this result is due to the similarity of the proteins’ native structure and the lack of a counter-selection round during the SELEX process. The error bars reflect standard deviations derived using at least three independently fabricated sensors. (B) The sensor Figure 5. Our EAB sensors can fully support the detection of the is rapid and can respond to the protein target in less than 20 s upon SARS-CoV-2 S protein and the RBD directly in biological fluids, the addition of a clinically relevant concentration of the SARS-CoV-2 demonstrating all their potential to perform frequent testing. When S protein (top) and the RBD (bottom). The electrochemical signal the EAB sensors are challenged in (A) fetal bovine serum (FBS) and was collected at 300 Hz (red) and 5 Hz (black) square wave (B) 50% artificial saliva, they can detect both viral protein targets, frequencies. The binding curves and kinetic experiments were displaying measurements with picomolar precision. The error bars obtained in PBS buffer, 2 mM MgCl2, pH 7.4 at 25 °C, using square reflect standard deviations derived using at least three independently wave voltammetry (SWV). fabricated sensors. discriminate the different RBDs from SARS-CoV-2 and SARS- selected these biological fluids because the former is routinely CoV-1 through a single measurement at 300 Hz or through a used for COVID-19 diagnostics (e.g., PCR, antigenic tests, and double detection at both frequencies. Therefore, our sensing cellular culture) due to the presence of the high concentration platform displays all its potential to discriminate different S of viral particles,64,65 whereas the latter allows studying the proteins variants and this could be improved only by the EAB sensor response in extremely complex media. When the introduction of counter-selection steps during the selection of sensor is employed in undiluted FBS (Figure 5A), we observe the aptamer to further improve its specificity. maximum signal gain values of −9.2 ± 0.9 and +16.0 ± 1.3% at The sensor response is so fast that can recognize the SARS- 5 and 300 Hz, respectively, and KD values of 300 ± 100 and CoV-2 RBD target in seconds, demonstrating its ability to 800 ± 200 pM at 5 and 300 Hz, respectively. Although the support point-of-care (PoC) sensing strategies. To evaluate the estimated affinities are lower than those obtained using the resolution time of the sensor, we exploited the ability of EAB buffer solution, the sensor’s dynamic range still covers the sensors to perform high-frequency measurements. We clinically relevant range.34,48,50 We believe that this difference collected the SWV signals at both frequencies (5 and 300 arises from the fouling effect, which reduces the formation of Hz), and after achieving a stable baseline (approximately after electrostatic and van der Waals interactions between the target 5 min), the sensor was challenged with 10 nM S protein and the sensing surface.57 Then, the sensor was tested in 50% (Figure 4B, top). The sensor responds within 15 s from the artificial saliva (Figure 5B). Specifically, at 5 Hz, the sensor still addition of the target, which corresponds to the required time responds to the target with a maximum signal gain of −7.3 ± to collect the two voltammograms. To further demonstrate the 0.2% and an estimated KD of 28.2 ± 5.0 pM. At 300 Hz, we rapidity of the binding kinetics, we challenged the sensor also estimated a maximum signal gain of +5.0 ± 0.2% and an with the RBD protein. Again, the sensor promptly responded estimated KD of 14.9 ± 2.0 pM. Motivated by the results, we to the injection of the RBD protein within 15 s, which further characterize our EAB sensor in undiluted artificial saliva demonstrates that the sensor’s response is not affected by the (Figure S6) to understand if it could be translated to work in target’s size (Figure 4B, bottom). real saliva samples. Using this condition, the sensor is still able Because the signal transduction mechanism is based on the to detect the presence of the protein target in its clinical range; binding-induced conformational change of the recognition however, it displays a lower signal gain. We believe that the element, EAB sensors work well when used directly in lower response is ascribed to the effect of the matrix on the E https://doi.org/10.1021/acssensors.1c01222 ACS Sens. XXXX, XXX, XXX−XXX ACS Sensors pubs.acs.org/acssensors Article aptamer’s conformational change. This suggests the need for strength of the matrix) allowed the detection of the target an optimization study for its next employment for clinical or protein in artificial saliva. precommercial applications. Although biological fluids such as saliva could have an impact on the performance of the EAB sensor, the overall data demonstrate that our developed sensor ■ ASSOCIATED CONTENT * Supporting Information sı could be used as a diagnostic tool for the rapid detection of the The Supporting Information is available free of charge at SARS-CoV-2 S protein. https://pubs.acs.org/doi/10.1021/acssensors.1c01222. ■ CONCLUSION In this paper, we described the development of a new EAB Materials and methods; binding and melting curves for optically labeled aptamer variants; frequency map; and calibration curves performed in 100% saliva of the EAB sensor able to detect the SARS-CoV-2 S protein rapidly and sensor (PDF) efficiently in biological fluids. First, we characterized the binding activity of the selected aptamers for the S protein using fluorescence spectroscopy. Through the use of optically labeled ■ AUTHOR INFORMATION Corresponding Author variants, we demonstrate that the aptamers can support a Arben Merkoçi − Institut Català de Nanociència i binding-induced conformational change mechanism, making Nanotecnologia (ICN2), Campus UAB, Bellaterra 08193 them ideal candidates to support EAB sensing platform. The Barcelona, Spain; CSIC and the Barcelona Institute of resulting EAB sensor showed comparable bioanalytical Science and Technology (BIST), 08036 Barcelona, Spain; performance in comparison to other methods using antibod- Institucio′ Catalana de Recerca i Estudis Avançats (ICREA), ies34,50,66 and aptamers67,68 for the detection of SARS-CoV-2 08010 Barcelona, Spain; orcid.org/0000-0003-2486- antigens. Specifically, using the 1C variant, we detected 8085; Email: arben.merkoci@icn2.cat picomolar levels of the S protein in buffer, serum, and 50% artificial saliva. The specificity of the results are also very Authors promising, indicating the ability of the EAB sensor to Andrea Idili − Institut Català de Nanociència i discriminate between similar targets (RBD portions of other Nanotecnologia (ICN2), Campus UAB, Bellaterra 08193 coronaviruses) and other proteins, besides being able to detect Barcelona, Spain; orcid.org/0000-0002-6004-270X the target in undiluted serum and artificial saliva. Finally, the Claudio Parolo − Institut Català de Nanociència i quick binding kinetics of the aptamer and its single-step Nanotecnologia (ICN2), Campus UAB, Bellaterra 08193 operation allow the detection of the target in 15 s, making our Barcelona, Spain; Barcelona Institute for Global Health, EAB sensor ideal to support high-frequency testing at the point 08036 Barcelona, Spain; orcid.org/0000-0001-9481- of care. Undoubtedly, other technologies that have been used 4408 for decades (e.g., LFA and ELISA)17,69 are still far ahead in Ruslán Alvarez-Diduk − Institut Català de Nanociència i terms of their application and commercial availability; Nanotecnologia (ICN2), Campus UAB, Bellaterra 08193 however, with this work, we want to show the potential of Barcelona, Spain; orcid.org/0000-0002-9876-1574 EAB sensing as a valid alternative to the current PoC tests. Complete contact information is available at: Additionally, their ability to support calibration-free and dual- https://pubs.acs.org/10.1021/acssensors.1c01222 reporter approaches70,71 (i.e., allowing the direct quantification of the target) and their versatility to be coupled with mobile Notes phones or portable electrochemical setups could represent an The authors declare no competing financial interest. additional tool to achieve real-time epidemiology.26,34 The secondary goal of this study is to help the community during the design and development of aptamer-based sensors, ■ ACKNOWLEDGMENTS ICN2 was funded by the CERCA programme, Generalitat de specifically (but not limited to) those relying on the binding- Catalunya. ICN2 was supported by the Severo Ochoa Centres induced conformational change. For example, looking in of Excellence programme and funded by the Spanish Research particular at the aptamer characterization, our results clearly Agency (AEI, grant no. SEV-2017-0706). The authors reinforce the need to introduce in the SELEX process both acknowledge Consejo Superior de Investigaciones Científicas counter-selection steps and the use of buffers with different (CSIC) for the project “COVID19-122” granted in the call ionic strengths. The former is crucial to select aptamers that “Nuevas ayudas extraordinarias a proyectos de investigación en specifically bind through a conformational change the target el marco de las medidas urgentes extraordinarias para hacer protein even in the presence of similar contaminants. For frente al impacto económico y social del COVID-19 (Ayudas example, the possibility to discriminate between the RBD CSIC-COVID-19)”. A.I. was supported by PROBIST post- portions of SARS-CoV-1 and SARS-CoV-2 would allow the doctoral fellowship funded by the European Research Council precise diagnosis of similar yet different diseases, requiring (Marie Skłodowska-Curie grant agreement no. 754510). different treatments and containing actions.72 The latter is R.A.D. received financial support from the EU Graphene important to guarantee the binding to the target in natural Flagship Core 3 Project (No. 881603). The authors also thank conditions, as it could be in a highly ionic strength Salvador Bartolomé Piñol, PhD and the Laboratori Lumines- environment such as saliva. Nonetheless, even with a cèn cia i Espectroscòp ia Biomolèc ules (LLEB) of the suboptimal selection, the aptamers we tested showed excellent Autonomous University of Barcelona (UAB). binding performance as bioreceptors in an EAB sensor. 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