Volume 1 Number 5 Sensors & September 2022 Pages 869–1090 Diagnostics rsc.li/sensors ISSN 2635-0998 PAPER Maria Soler et al. Rapid and direct quantification of the SARS-CoV-2 virus with an ultrasensitive nanobody-based photonic nanosensor Sensors & Diagnostics This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Rapid and direct quantification of the SARS-CoV-2 Cite this: Sens. Diagn., 2022, 1, 983 virus with an ultrasensitive nanobody-based photonic nanosensor Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. Gisela Ruiz-Vega,a Maria Soler, *a M. Carmen Estevez,a Patricia Ramirez-Priego, a Martalu D. Pazos,a María A. Noriega,b Yago Margolles,c Clara Francés-Gómez,d Ron Geller,d Giulia Matusali,e Francesca Colavita,e Antonino di Caro,†e José M. Casasnovas, b Luis Angel Fernándezc and Laura M. Lechuga a The coronavirus (CoV) disease 2019 (COVID-19) is expected to become endemic in the coming years, meaning that the worldwide society shall be prepared to routinely manage the highly contagious respiratory SARS-CoV-2. A rapid and early diagnosis of the SARS-CoV-2 infection is crucial for controlling the spread of the disease, interrupting the transmission chain, and providing timely medical attention to patients. We introduce an innovative nanophotonic biosensor for the quantitative detection of viral particles in less than 20 minutes total assay time. The nanosensor, based on the bimodal waveguide (BiMW) interferometric technology, has been functionalized with novel bioengineered nanobodies (Nb) targeting the SARS-CoV-2 receptor-binding domain (RBD). Our approach relies on the direct capture of the viral particles, and the optimized methodology allows the detection of the SARS-CoV-2 virus with outstanding sensitivity, below 200 TCID50 per mL, being able to provide accurate viral load determination within a broad dynamic range (102–106 TCID50 per mL). Both the nanobodies and the sensor nanotechnology can Received 9th May 2022, be produced at a large scale with highly-efficient cost-effective procedures, and they are being integrated Accepted 14th June 2022 into a user-friendly point-of-care device for multiplexed and decentralized operation. The implementation of this unique biosensor in primary care assistance, hospitals, pharmacies, or private laboratories could DOI: 10.1039/d2sd00082b greatly aid in the relief and descongestion of the sanitary systems and the clinical and social management rsc.li/sensors of COVID-19. Introduction that enable rapid, sensitive, and accurate detection of respiratory viral infections at the point of need, being the The outbreak of the Coronavirus Disease 2019 (COVID-19) primary and most effective way to interrupt the transmission and its pandemic spread have evidenced the urgent need of chain and to provide timely medical attention to patients. developing and implementing advanced diagnostic systems Currently, clinical diagnosis of SARS-CoV-2 (i.e., Severe Acute Respiratory Syndrome Coronavirus 2) infection is performed a through nucleic acid amplification tests based mainly on the Nanobiosensors and Bioanalytical Applications group (NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST, and CIBER-BBN, reverse transcription-polymerase chain reaction (RT-PCR). Bellaterra, 08193, Barcelona, Spain. E-mail: maria.soler@icn2.cat PCR-based assays are undoubtedly one of the most robust b Department of Macromolecular Structure, Centro Nacional de Biotecnología, and precise existing techniques for disease diagnosis, offering Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus outstanding sensitivity and specificity (96–98% and 99%, Cantoblanco UAM, 28049 Madrid, Spain c respectively), standardized procedures, and a relatively facile Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus adaptation to emerging pathogens or biomarkers.1,2 Cantoblanco UAM, 28049 Madrid, Spain However, its out-of-the-lab deployment as a point-of-care d Institute for Integrative Systems Biology (I2SysBio), Universitat de Valencia-CSIC, (POC) testing system is still a major limitation, mostly 46980, Valencia, Spain restricted by the sample processing needs (e.g., RNA e National Institute for Infectious Disease “L. Spallanzani” IRCCS, Via Portuense extraction) and the sophisticated multi-step analytical 292, 00149, Rome, Italy † Current affiliation: Saint Camillus International University of Health Sciences, process3,4 or limitations in the field deployment of still quite via di Sant'Alessandro, 8, 00131 Roma, Italy; IRCCS Sacro Cuore Don Calabria complex and expensive equipment. As an alternative, Hospital, via Don A. Sempreboni 5, 37024, Negrar di Valpolicella (Verona), Italy. immunochromatographic lateral flow assays (LFA) have been © 2022 The Author(s). Published by the Royal Society of Chemistry Sens. Diagn., 2022, 1, 983–993 | 983 View Article Online Paper Sensors & Diagnostics proposed and broadly employed as a rapid qualitative ideal technology, performing ultrasensitive analysis in a diagnostic test for COVID-19. LFAs targeting viral antigens label-free and real-time format.7–9 Latest research on label- have been developed for direct patient use, being affordable free photonic biosensors has demonstrated their excellent and not requiring any trained sanitary personnel or capabilities for detection and identification of respiratory This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. specialized equipment, delivering a clear yes/no response in virus infections, such as influenza, respiratory syncytial virus, less than 15 minutes. The convenience of such rapid and coronaviruses.7 For SARS-CoV-2, few studies have been diagnostic tests has been proven, especially in late pandemic recently published fully addressing the label-free optical stages for general population screening, and risk biosensing of viral particles,10–12 viral RNA,13 or viral minimization in mass-attendance events. However, most antigens.14 Besides, photonic biosensors have been LFAs suffer from insufficient sensitivity for early diagnosis, developed for serological assays through the detection and showing false-negative results at the initial stages of the quantification of COVID-19 antibodies, although only a few Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. infection (65–70% sensitivity, 97–99% specificity).5,6 Besides, have been clinically validated.15,16 Nonetheless, the sensitivity the majority of COVID-19 LFAs are purely qualitative and levels achieved so far for the direct detection of SARS-CoV-2 cannot determine the viral load, which would aid in patient infection have not reached yet the clinical standards set by clinical management. PCR-based assays, or they involve costly and sophisticated Biosensors hold great promise for attaining accurate, instrumentation and bioreagents making them unsuitable rapid, and simple diagnosis of COVID-19. Among the for eventual implementation for POC mass testing. different sensor modalities, photonic biosensors based on We present an innovative nanobiosensor for the direct the evanescent field principle have been positioned as an diagnosis of COVID-19 that provides ultrasensitive detection Fig. 1 (a) Illustration of the bimodal waveguide (BiMW) biosensor for direct detection of the whole SARS-CoV-2 virus using bioengineered nanobodies (Nb) as recognition elements. (b) Schematics of the BiMW interferometer sensing principle. (c) Photograph of the BiMW sensor chip – 1 Euro coin is included for dimension comparison. (d) Photograph of the laboratory set-up for BiMW biosensing measurements. 984 | Sens. Diagn., 2022, 1, 983–993 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Sensors & Diagnostics Paper of the SARS-CoV-2 virus over a broad range of concentrations VHH domains derived from heavy chain-only antibodies (102–106 viral particles per mL), by direct and specific capture (hcAb) naturally found in camelids (e.g., dromedaries, llamas, of viral particles, without any additional amplification step, alpacas). They are the smallest antigen-binding Ab fragments delivering quantitative data in less than 20 minutes sample- known (ca. 14 kDa) and can be obtained by recombinant This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. to-result time, and importantly, with potential scalability for expression in bacteria, yeasts, and/or mammalian cell affordable and efficient point-of-care testing. The biosensor systems.28 Despite their small size, they have identical is based on a bimodal waveguide (BiMW) interferometer, a affinity and antigen specificity to conventional Ab molecules powerful silicon photonics sensor nanotechnology that with heavy chain (i.e., immunoglobulin G, IgG). Nb have exploits the label-free evanescent wave sensing principle additional properties such as an enhanced stability to (Fig. 1).17,18 The BiMW technology employs a single straight thermal and chemical denaturation, higher solubility, waveguide where light, at visible wavelengths, is confined resistance to proteolysis, and the ability to bind to small Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. and propagates, generating an evanescent field that protein cavities such as the active centers of enzymes and penetrates hundreds of nanometers in the adjacent dielectric conserved inner regions on pathogen surfaces, which remain external medium. This evanescent field is highly sensitive to hidden to human antibodies to avoid immune minute changes of the refractive index (RI), such as those neutralization.29,30 These superior characteristics provide caused by a biointeraction event. In particular, the bimodal unique advantages in the use of Nb for diagnostic waveguide allows the confinement of two light modes of the applications and especially for nanobiosensor same polarization, whose respective evanescent fields behave development.31,32 Moreover, they can be expressed as a differently to RI changes, resulting in different propagation monovalent molecule, or as protein fusions to other Nb and/ and thus in an interferometric phase shift (Δφ) that can be or protein domains (e.g., Fc domain of IgG), producing readily monitored. This design involves significantly less bivalent and multivalent molecules with mono-, bi-, or multi- complex fabrication processes than conventional specific binding capabilities.30,33 Herein, we employed hcAb interferometric configurations that require a split waveguide containing previously selected VHH domains with high in two arms (e.g., Mach–Zehnder interferometers) and affinity for the RBD fusioned to the Fc region of human IgG1, facilitates the integration in small footprint optofluidic which have shown excellent recognition for many SARS-CoV-2 devices, without sacrificing their remarkable sensitivity.19 In variants.27 We have compared the performance of the device fact, the BiMW biosensor stands out among the different incorporating the hcAb with commercial Ab targeting the label-free photonic sensors due to its extraordinary RBD, and carefully optimized the conditions that allow the refractometric resolution (i.e., 10−7–10−8 refractive index detection of the SARS-CoV-2 virus. units, RIU), which is between one and two orders of Our results represent the first report demonstrating the magnitude better than the benchmarked surface plasmon feasibility of employing nanophotonic BiMW sensors for resonance (SPR) systems.20 These nanophotonic sensor chips direct SARS-CoV-2 virus detection and quantification in a are currently fabricated with well-established simple, direct way, reaching limits of detection (LOD) of a microelectronics processes and we have implemented them few hundreds of viruses (viral load ≈ 100–200 virus per mL). in biosensor prototypes, demonstrating outstanding The assay is completed in a few minutes, thus showing performance for the direct analysis of proteins and nucleic promising performance to be eventually implemented at acids, with detection limits in the aM range,21–23 and also for decentralized settings in the healthcare system. large pathogenic bacteria, achieving direct detection of only a few cells per milliliter (i.e., 30–50 CFU mL−1).24,25 Experimental One of the most critical challenges for the direct detection Chemical and biological reagents of an emerging and previously unknown virus is the availability of appropriate bioreceptors.7,26 Many antibodies Triethoxysilane polyethylene glycol carboxylic acid (silane- targeting the spike (S) protein of SARS-CoV-2, or its receptor PEG-COOH, 600 Da) was supplied by Nanocs (New York, US). binding domain (RBD), have been produced and Reagents for carboxylic acid activation, (N-(3- commercialized in this critical task. However, the laborius dimethylaminopropyl)-ethylcarbodiimide hydrochloride and costly manufacturing process poses important (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS)), were limitations for their use in standard diagnostics, especially in provided by Sigma-Aldrich (Steinheim, Germany). Chimeric terms of reproducibility and specificity. To demonstrate the monoclonal antibodies (mAb) against RBD (anti-RBD mAb), potential of our technology to detect a small number of virus recombinant S1 and RBD proteins were purchased from Sino entities directly in patient samples, we have designed a Biological Europe GmbH (Germany). Human C-reactive detection strategy based on the direct capture of the SARS- protein (CRP) was acquired from BBI Solutions (UK). Viral CoV-2 virus through specific bioengineered receptors transport media (VTM) were acquired at Deltalab (Spain). designed for this purpose. We have combined the high- performance biosensor nanotechnology with high-affinity Production of Nb-derived hcAb (Nb–Fc) nanobodies (Nb) recently produced against the receptor- The hcAb that contain the Nb fused to the Fc of IgG1 were binding domain (RBD) of the S protein.27 The Nb are single expressed upon DNA transfection with the corresponding © 2022 The Author(s). Published by the Royal Society of Chemistry Sens. Diagn., 2022, 1, 983–993 | 985 View Article Online Paper Sensors & Diagnostics plasmid vector of suspension human embryonic kidney BiMW interferometric sensor chip and biosensor device (HEK-293F) mammalian cell, reported previously.27 The The BiMW sensors are fabricated at a wafer-scale through secreted hcAb were purified from culture supernatants standard microelectronics technology processes in the Micro using protein A affinity columns (Cytiva) and further and Nanofabrication Clean Room of the National This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. purified by size exclusion chromatography (SEC) with a Microelectronics Center (IMB-CNM-CSIC, Barcelona, Spain). Superdex75 (10/300) column (Cytiva) in HBS (20 mM The photonic chip (1 cm width × 3 cm length) contains an array HEPES, 150 mM NaCl, pH 7.4). of 20 individual straight rib waveguides (rib height 1–3 nm) (Fig. 1c). Two differentiated regions are generated through the Production of VSV-ΔG-S pseudotyped virus creation of a step junction that allows first the propagation of the fundamental mode (waveguide core thickness of 150 nm) A vesicular stomatitis virus pseudotyped with the Wuhan and later in the second section the propagation of the Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. variant of the SARS-CoV-2 spike protein (VSV-ΔG-S) was fundamental and first-order modes (waveguide core thickness produced as previously described34 at the I2SysBio facilities of 340 nm) (see Fig. 1b). In a section of the bimodal rib (University of Valencia-CSIC, Spain). Similarly, a control VSV waveguide, the cladding is etched to generate the sensing area pseudotyped with its own glycoprotein (VSV-ΔG-G) was (15 × 0.05 mm2) where the evanescent field of the guided light produced by transfection with pMD2.G (Addgene plasmid can probe the surrounding dielectric environment. The chosen 12 259). The virus was concentrated by centrifugation at material of the waveguide core is silicon nitride (Si3N4) because 50 000 × g for 4 hours, and resuspended in PBS at moderate/ of its high refractive index (ncore = 2.00), high density, and high titers (i.e., 106 focus forming units per mL (FFU mL−1)). chemical inertness to ion species, oxygen, or moisture permeation. The core is confined by an upper and lower Production of the UV-inactivated SARS-CoV-2 viral stock cladding made of silicon oxide (SiO2) with a lower refractive index (ncladd = 1.46) to ensure the guiding of light through the SARS-CoV-2 stock preparation was performed in the biosafety core with minimal loss of energy. The BiMW interferometric level 3 (BSL-3) laboratory at the National Institute for biosensor system employs a low-power polarized laser diode at Infectious Diseases (INMI) in Rome, Italy. Vero E6 cells (ATCC a visible wavelength (λ = 660 nm, P = 120 mW) as a light source Number CRL-1586™) were used and maintained in Modified coupled into the rib waveguide, and a two-sectional photodiode Eagle's Medium (MEM) containing 10% heat-inactivated fetal as a detector for the interferometric signal readout (Fig. 1d). serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin The optical system is mounted on a Peltier thermoelectric solutions, at 37 °C in a humidified atmosphere of 5% CO2. cooler, to assure thermal stability with an accuracy of 0.01 °C. Vero E6 cells were infected with a SARS-CoV-2 Wuhan-D614G The system incorporates two photodiode amplifiers to read the strain (lineage B.1 – clade G), isolated in Italy in March 2020 signal of both sections of the photodiode and an acquisition (GISAID accession number: EPI_ISL_568579, EVAG Ref-SKU: board is used for setup controlling. Data processing is 008 V-04005). Specifically, cells were exposed to SARS-CoV-2 performed with a customized software interface that isolate at a multiplicity of infection (MOI) of 0.01 in MEM incorporates a recently developed optical phase modulation without FBS for 1 hour at 37 °C/5% CO2. At the end of the method to transform the complex interferometric signals into a adsorption period, the inoculum was replaced with fresh linear signal (Δφ), directly proportional to the change of the MEM not containing FBS and incubated at 37 °C/5% CO2. refractive index occurring at the sensor area which allows real- After seventy-two hours post-infection, a cytopathic effect time monitoring.20 The sensor chip is integrated with a five- (CPE) was visible on about 80–90% of the cell culture and channel polydimethylsiloxane (PDMS) microfluidic system viral preparation was performed at −80 °C. Following (channel dimensions = 1.25 mm wide × 500 μm height), for thawing, a clarified cell supernatant was exposed to continuous flow and washing of the samples. The fluidic ultraviolet (UV) radiation (λ 254 nm) for 15 minutes on ice. At system also includes a syringe pump to guarantee a continuous the end of the UV-treatment, the supernatant was aliquoted flow rate of a running buffer and a 6-port injection valve and stored at −80 °C. An aliquot of SARS-CoV-2 preparation connected to a loop (100 μL) that allows the sequential loading not exposed to UV was used as a control of the inactivation and injection of different samples and solutions. process and to calculate the virus stock titer according to the method of Reed and Muench and expressed as 50% tissue culture infectious dose (TCID50 per ml).35 The virus stock Sensor surface biofunctionalization titer was: 107 TCID50 per mL. The presence of residual Before surface functionalization, the sensor chips are infectivity in UV-exposed samples was assessed by infecting consecutively sonicated for 5 min in acetone, ethanol, and the Vero E6 cell culture. After five days post-infection, no CPE Milli-Q water, and 10 min in methanol/HCl 1 : 1 (v/v), to was observed. To further exclude residual infectivity, the remove organic contamination. The sensor chips are then related cell supernatant was back-titrated by limiting the rinsed with water and dried with a stream of nitrogen. A layer dilution assay on Vero E6 cells; no virus growth was detected. of active hydroxyl groups is generated onto the sensor surface The UV-inactivated SARS-CoV-2 stock was shipped to ICN2 using oxygen plasma for 5 min at 45 sccm gas flow, followed (Barcelona, Spain) at controlled temperature. by immersion in a 15% HNO3 solution at 75 °C for 25 min. 986 | Sens. Diagn., 2022, 1, 983–993 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Sensors & Diagnostics Paper After rinsing with water and drying under a N2 flow, the (i.e., around 100 nm diameter compared with considerably sensor chip is immediately immersed and incubated in a 25 larger bacteria, as 1–3 μm). Commercial antibodies were mg mL−1 silane-PEG-COOH solution in ethanol/water 95 : 5 initially selected to assess the preliminary conditions for sensor (v/v) for 2 h at 4 °C. After the incubation, the sensor chip is chip biofunctionalization, antigen detection, and efficient virus This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. rinsed with ethanol and water and dried with an N2 stream. capture. We employed a commercial monoclonal antibody A final curing step is performed by introducing the sensor targeting the receptor-binding domain of the spike protein of chips into a glass container inside a conventional autoclave SARS-CoV-2 (i.e., anti-RBD mAb). Monoclonal antibodies were for 90 min at 120 °C at a pressure of 1.5 bar. For bioreceptor immobilized on the BiMW sensor surface via carbodiimide immobilization, the silanized sensor chip is placed on the crosslinking to a carboxyl-functional silanized surface (Fig. 2a). experimental setup for the covalent attachment of the The silane employed for sensor surface functionalization antibodies. The carboxyl groups are activated by flowing a includes a polyethylene glycol (PEG) chain (600 kDa) that helps Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. solution of 0.2 M EDC/0.05 M sulfo-NHS in MES buffer (0.1 prevent and minimize undesired non-specific adsorption M, pH 5.5) at 20 μL min−1 over the sensor surface. The events. Affinity toward SARS-CoV-2 was first evaluated through antibody solution at an optimum concentration is then the direct and label-free detection of the external viral antigens injected at a flow rate of 10 μL min−1. The remaining (recombinant S1 and RBD proteins), employing a non-related unreacted activated groups are deactivated with an protein (C-reactive protein) as a negative control for specificity ethanolamine solution (1 M, pH 8.5) for 2 minutes at 20 μL assessment. As can be seen in Fig. 2b, the mAb showed efficient min−1. Milli-Q water is used as the running buffer during the detection of both viral antigens, with limits of detection immobilization process. determined in the range of ng mL−1, and null cross-reactivity with the negative control. Besides, the robustness and stability Direct immunoassay performance and data analysis of the biofunctionalized surface were confirmed by flowing a Once the sensor chip surface is biofunctionalized, the running high-pH solution (NaOH 20 mM), efficiently regenerating the buffer is replaced by the selected detection buffer (PBST 0.1% recognition interface by removing the captured viral antigens, for protein and pseudovirus detection, and HEPES 25 mM for without detaching or damaging the immobilized antibodies up SARS-CoV-2 detection). Purified samples of different to 12 measurement cycles (data not shown). concentrations of proteins, pseudovirus, or UV-inactivated The viability of this biofunctionalization protocol for the SARS-CoV-2 are injected into the system at a constant flow rate detection of viral particles was further assessed employing a of 10 μL min−1. Biosurface regeneration was performed after SARS-CoV-2 pseudovirus, a non-replicative virus (i.e., every detection with NaOH 10 mM. All experiments for virus vesicular stomatitis virus, VSV) specifically engineered to detection were carried out in the ICN2 BSL2 facilities with express the S protein of SARS-CoV-2 in its outer membrane approval of the UAB Biosafety Committee (HR-599-20). Data (VSV-ΔG-S). A VSV pseudotype virus expressing its were analyzed using Origin 8.0 (OriginLab, MA, US) and corresponding glycoprotein (G) and lacking S protein was GraphPad Prism 8 (GraphPad Software, CA, US). Calibration employed as a negative control for specificity studies (i.e., curves were plotted as the mean and standard deviation (mean VSV-ΔG-G). These pseudoviruses can mimic the SARS-CoV-2 ± SD) of the acquired biosensor response (Δφ) versus the target virus in terms of size and antigen expression and can be concentration. The phase variation (Δφ) considered was the safely used in BSL2 facilities to evaluate the BiMW one observed after signal stabilization, once the whole sample biosensor because they do not encode an entry glycoprotein had completely passed through the sensor chip (i.e., t ≈ 900 s). in their genome, and, therefore, can only perform a single The data were fitted to a two-site specific binding model curve. round of infection. Serial dilutions of the VSV-ΔG-S The LOD, defined as the smallest concentration pseudovirus (1 × 102–1 × 105 FFU mL−1) were directly distinguishable from the blank, and LOQ, defined as the analyzed with the BiMW sensor functionalized with specific minimum concentration that can be reliably detected and anti-RBD mAb, demonstrating the potential of our biosensor quantified, were determined as the concentration technology for rapid detection and quantification of whole corresponding to three or ten times the standard deviation viral particles with excellent sensitivity (Fig. 2c and d). Also, (SD) of the baseline of the sensor signal, respectively. the assay selectivity was confirmed by flowing a non- modified VSV pseudovirus (VSV-ΔG-G) at high Results and discussion concentrations, showing zero signal response (red curve in Fig. 2d). With the standard calibration curve obtained with Evaluation of the BiMW biosensor for direct capture and the SARS-CoV-2 pseudovirus and the commercial detection of viral particles monoclonal antibody, we determined the limit of detection The BiMW biosensor has previously demonstrated its potential (LOD) and limit of quantification (LOQ) of the biosensor for direct, label-free detection of target biomolecules of different assay at 598 FFU mL−1 and 1119 FFU mL−1, respectively. nature, like proteins and small molecules,20,36,37 DNA and RNA These sensitivity values already show the excellent sequences,38–40 and also for the direct capture of bacteria.24,25 performance and potential of our biosensor for COVID-19 These results set the basis for exploring the use of such a device diagnosis, with common levels of SARS-CoV-2 in patients for the detection of viruses, much smaller entities than bacteria found between 104 and 106 viral particles per mL.41 © 2022 The Author(s). Published by the Royal Society of Chemistry Sens. Diagn., 2022, 1, 983–993 | 987 View Article Online Paper Sensors & Diagnostics This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. Fig. 2 (a) Representative real-time sensorgram of the anti-RBD mAb (20 μg mL−1) three-step immobilization procedure through carbodiimide chemistry. (b) Calibration curves for direct detection of recombinant SARS-CoV-2 viral antigens (S and RBD protein, orange and blue, respectively), including negligible signals of the negative control (C-reactive protein, CRP, red). (c) Standard calibration curve for direct detection of the SARS- CoV-2 pseudovirus (i.e., VSV-ΔG-S), obtained with triplicate measurement of serial virus sample dilution (100–105 FFU mL−1). (d) Representative sensorgrams of the detection of different SARS-CoV-2 pseudovirus concentrations (blue curves), and sensor response for the non-modified pseudovirus (VSV-ΔG-G) as the specificity control (red curve). We also studied the effect of different virus inactivation by the antibodies – taking the pseudovirus as a model. This methods on the integrity of the virus – and its recognition was a necessary step before attempting the detection of Fig. 3 (a) Biosensor response for the detection of the VSV-ΔG-S pseudovirus (1 × 105 FFU mL−1) after different inactivation treatments: non- treated (blue), UV treatment at λ 254 nm for 15 min (purple), heat at 56 °C for 30 min (yellow), and heat at 95 °C for 10 min (orange). Each column represents the mean and standard deviation of 3 replicates. (b) Transmission electron microscopy (TEM) images of the SARS-CoV-2 virus inactivated through UV treatment (left), heat at 56 °C for 30 min (middle), and heat at 95 °C for 10 min (right). 988 | Sens. Diagn., 2022, 1, 983–993 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Sensors & Diagnostics Paper SARS-CoV-2 in the BSL2 facilities, restricted to only Bioengineered nanobodies (Nb–Fc) for SARS-CoV-2 detection inactivated, non-infectious specimens. Three main inactivation procedures commonly employed were tested With these initial conditions for biofunctionalization and virus with the non-infectious pseudovirus samples: exposure to detection, we further evaluated the sensitivity we could achieve This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ultraviolet (UV) radiation (15 min, λ 254 nm), mild long with high-quality, high-affinity nanobodies specifically heat treatment (30 min, 56 °C), and harsh short heat designed and selected for binding to the RBD. The biomolecule treatment (10 min, 95 °C). The sensor response of the VSV- used for virus detection was a fusion protein between the VHH ΔG-S virus sample after each inactivation treatment was domain encoding the nanobody called 1.26 and the constant compared to the non-treated sample (Fig. 3a). As expected, fraction (Fc) of human IgG1, which has high structural heat treatment drastically decreased the sensor signal similarities with conventional immunoglobulin,27 facilitating corresponding to the detection by the immobilized analogous biofunctionalization outcomes. The bioengineered Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. antibodies, while UV radiation did not have any significant 1.26 Nb–Fc fusions were immobilized on the silanized BiMW effect on the biosensor assay. In fact, the heating of virus sensor surface following the carbodiimide chemistry procedure samples disrupts the membrane structure and denaturalizes previously described and optimized for commercial the proteins, hampering the direct recognition of the viral monoclonal antibodies (Fig. 4a). We first compared the direct antigens with antibodies. In contrast, the UV treatment is detection of the VSV-ΔG-S pseudovirus in analog experiments intended to only affect the genomic material, seemingly to the ones performed with commercial monoclonal unaltering the structure of the viral particles (Fig. 3b). antibodies. The standard calibration curve was obtained, Fig. 4 (a) Representative sensorgram of the anti-RBD Nb–Fc immobilization procedure through carbodiimide chemistry. (b) Standard calibration curve for direct detection of the pseudovirus (i.e., VSV-ΔG-S), obtained with triplicate measurement of serial virus sample dilution (100–105 FFU mL−1). (c) Standard calibration curve for direct detection of the UV-inactivated SARS-CoV-2 virus, obtained with triplicate measurement of serial virus sample dilution (100–106 TCID50 per mL). Inset shows a zoom-in of the low-range virus concentrations (0–104 TCID50 per mL). (d) Representative real-time sensorgrams for the detection of the SARS-CoV-2 virus at low concentrations (100–5000 TCID50 per mL). © 2022 The Author(s). Published by the Royal Society of Chemistry Sens. Diagn., 2022, 1, 983–993 | 989 View Article Online Paper Sensors & Diagnostics reaching a LOD and LOQ of 162 FFU mL−1 and 727 FFU mL−1, Biosensor assay performance on sample collection media respectively (Fig. 4b). This detection limit is more than 3 times better than the value achieved with the commercial Progressing towards the clinical validation of the nanobody- monoclonal antibody, proving that the bioengineered 1.26 Nb– based photonic biosensor technology for COVID-19 This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fc, which showed excellent affinity (KD = 80 pM) for the RBD, diagnostics, we have further evaluated the direct detection of can provide enhanced performance for diagnostics purposes. the SARS-CoV-2 virus under relevant clinical conditions. In On this promising basis, we addressed the direct capture, particular, we have assessed the detection of whole viruses in detection, and quantification of real SARS-CoV-2 viral a common viral transport medium (VTM) employed for particles employing bioengineered Nb as recognition diluting and preserving the nasopharyngeal swab specimens elements. An UV-inactivated Wuhan-D614G SARS-CoV-2 from sample collection until their arrival into the laboratory. isolate was used as the standard for biosensor calibration. Such VTMs have different compositions including Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. Fig. 4c shows the calibration curve obtained for SARS-CoV-2 preservatives, proteins, etc. that could adsorb onto the virus concentrations ranging from 100 to 106 TCID50 per biosensor surface, generating false-positive signals. The mL. As expected, compared to pseudoviruses, the sensor BiMW sensor is chemically modified with a thin layer of response and sensitivity for detection of real SARS-CoV-2 functional silane grafted with PEG chains, which are known greatly increased, with an outstanding LOD of 97 TCID50 to reduce nonspecific bindings and improve the antifouling per mL, and a LOQ of 339 TCID50 per mL. Such remarkable resistance. The ideal effect of this biofunctionalization values mean that we could directly visualize and quantify in method was confirmed by flowing a blank sample of the real-time, and in less than 20 minutes, the presence of VTM that resulted in a negligible signal (Fig. 5a). However, fewer than 100 infectious viral particles in a sample and the presence of different substances in the VTM can also over a wide range of concentrations (Fig. 4d). Our analytical affect the antigen–antibody recognition interaction as well as sensitivity is comparable to standard clinical PCR-based the dispersity of the viral particles in the sample. Such an techniques employed in hospitals (i.e., cycle threshold (Ct) effect could be observed by an increase in the variability values ranging between 30 and 15 approximately correspond among the samples and virus concentrations (data not to 102–105 TCID50) and is superior to most of the first rapid shown). To solve this, we included a specific additive to the LFAs for POC diagnosis.41–43 To further confirm the VTM (i.e., guanidine hydrochloride 1 M), which acts as a excellent performance of Nb as sensor biorecognition surfactant. Thereupon, we performed the calibration for the elements, we evaluated another bioengineered Nb (2.15 Nb– detection of the SARS-CoV-2 virus directly in the VTM. As Fc) showing similar RBD binding affinity (KD = 80 pM).27 seen in Fig. 5b, reliable detection and quantification of SARS- From the standard calibration curve performed for direct CoV-2 in VTM was confirmed, achieving a LOD of 178 detection of the SARS-CoV-2 virus, we determined a LOD of TCID50 per mL and a LOQ of 596 TCID50 per mL; slightly 87 TCID50 per mL and a LOQ of 303 TCID50 per mL (data superior to the ones achieved with purified samples but still not shown); the values very similar to the ones obtained well below the clinical cut-off for COVID-19 diagnostics (103– with 1.26 Nb–Fc. 104 TCID50 per mL). Fig. 5 (a) Sensorgram showing the response of the BiMW biosensor to the undiluted and untreated viral transport medium. (b) Standard calibration curve for direct detection of the SARS-CoV-2 virus spiked in VTM obtained with triplicate measurements of serial virus sample dilution (100–105 TCID50 per mL). Inset shows a zoom-in of the lower virus concentrations (0–104 TCID50 per mL). 990 | Sens. Diagn., 2022, 1, 983–993 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Sensors & Diagnostics Paper These preliminary results validate the potential of the a multiplexed BiMW sensor targeting parallel detection of BiMW nanobiosensor for the direct analysis of patient different respiratory viruses (e.g., influenza, human CoVs, etc.) nasopharyngeal samples, without requiring any amplification could be easily developed to allow one-step assay identification or pretreatment process – such as the ones generally of most common viral infections, greatly aiding in the control This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. performed for nucleic acid assays (e.g., RNA extraction and of these highly contagious endemic diseases. purification and RT-PCR), and delivering highly accurate and precise diagnostic results in less than 20 minutes total assay Author contributions time. To our knowledge, this is the first report demonstrating the application of a label-free photonic nanosensor for the GRV performed the experiments for biosensor optimization direct detection of the whole SARS-CoV-2 virus in their and evaluation for virus detection. MS and MCE supervised corresponding sample collection media. Previously reported the experiments, analyzed the data, and wrote the Open Access Article. Published on 14 June 2022. Downloaded on 3/7/2023 2:56:58 PM. studies, addressing the quantification of SARS-CoV-2 viral manuscript. PRP carried out the initial experiments for viral particles with label-free nanoplasmonic sensors,10,11 have antigen detection. LAF, JMC, YM and MAN designed, achieved detection limits in the range of 300 viral particles produced and purified the bioengineered Nb. RG and CFG per mL, which is two times higher than our BiMW produced and purified the SARS-CoV-2 pseudovirus samples. nanosensor (around 100 viruses per mL), their results were GM, FC, and ADC produced the UV-inactivated SARS-CoV-2 based on pseudoviruses as target samples and, in the case of samples. LML coordinates the project and supervised all Huang et al., performance is only demonstrated under experimental and discussion activities. All authors have read, standard buffer conditions. Both the biological assay revised, and approved the final version of the manuscript. approach and the analytical performance of our nanobiosensor have shown to be highly promising towards Conflicts of interest the implementation of new point-of-care technologies for There are no conflicts to declare. improved COVID-19 diagnostics. Acknowledgements Conclusions This work has been mainly funded by the H2020 Research Driven by the worldwide demand for point-of-care diagnostics and Innovation Program of the European Commission for COVID-19 testing, we introduce a pioneering nanophotonic (H2020-SC1-PHE-Coronavirus-2020, CONVAT No 101003544) biosensor that enables the quantitative analysis of the SARS- and by the European Commission – NextGenerationEU CoV-2 virus with an outstanding sensitivity – detection limits (Regulation EU 2020/2094), through CSIC's Global Health around 100 infectious viral particles per mL – in less than 20 Platform (PTI Salud Global – Iniciativa Estratégica de minutes total assay time. Our nanosensor, based on robust, Diagnóstico). The ICN2 is funded by the CERCA Program/ high-quality BiMW interferometers, is functionalized with novel Generalitat de Catalunya and supported by the Severo Ochoa bioengineered Nb against the SARS-CoV-2 RBD, and has proven Centers of Excellence program, funded by the Spanish superior affinity towards viral antigens and, importantly, can be Research Agency (AEI, grant SEV-2017-0706). Work in CNB- produced at a large scale with affordable and highly CSIC was funded by the Spanish Ministry of Science and reproducible techniques. The Nb–Fc employed as a sensor Innovation (MICIN) and the Spanish Research Council (CSIC) bioreceptor (1.26) has previously demonstrated good interaction under grants PIE-RD-COVID 19 (No 202020E079) and PTI affinity with different SARS-CoV-2 variants, therefore the same Salud Global REC_EU (No SGL 2103051, NextGenerationEU). behavior can be expected for biosensing applications. Work at I2SysBio was funded by Ayudas de concesión directa Furthermore, SARS-CoV-2 Nb–Fc can be easily bioengineered to a soluciones científico-innovadoras directamente relacionadas ensure efficient detection of different virus mutations and con la lucha contra la Covid-19 grant from the Generalitat variants. The biosensor immunoassay has been optimized and Valenciana, Spanish National Research Council grant CSIC- fully demonstrated for the direct capture and detection of the COV19-082/104, Fondo Supera Covid-19 grant BlockAce, and whole SARS-CoV-2 virus, eliminating the need for extraction and European Commission – NextGenerationEU to Ron Geller. amplification procedures, and providing a straightforward INMI received funds for this study from the Ministero della readout of not only the presence of infectious virus but also the Salute (Ricerca Corrente - linea 1), the European Commission viral load. The assay is equally effective and sensitive in viral – Horizon 2020 (EU project 101003544 – CoNVat) and the transport media compared with laboratory standard conditions, European Virus Archive – GLOBAL (grants no. 653316 and no. which reinforces the potential of performing direct analysis of 871029). We thank the Micro and Nanofabrication Clean collected nasopharyngeal swab samples. This new Room of the National Microelectronics Center (IMB-CNM- nanotechnology has unique potential to be implemented in CSIC, Barcelona, Spain) as a third-party partner in the healthcare and clinical practices. Our current work is dedicated CONVAT project for the fabrication of the BiMW photonic to integrating the sensor system in a compact and automated chips. We are very grateful to EPI Industries (Barcelona, device that will be installed in a hospital laboratory for clinical Spain) for its kind donation in supporting our research on validation directly with COVID-19 patient samples. 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