Biosensors and Bioelectronics 226 (2023) 115137 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Biomimetic nanoplasmonic sensor for rapid evaluation of neutralizing SARS-CoV-2 monoclonal antibodies as antiviral therapy Razia Batool a, Maria Soler a, *, Francesca Colavita b, Lavinia Fabeni b, Giulia Matusali b, Laura M. Lechuga a, ** a Nanobiosensors and Bioanalytical Applications Group (NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST, CIBER-BBN, Spain b National Institute for Infectious Disease “L. Spallanzani”, IRCCS, Rome, Italy A R T I C L E I N F O A B S T R A C T Keywords: Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the po­ Surface plasmon resonance tential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations COVID-19 recently approved by the European and American medicine agencies. However, a main bottleneck for their Immunotherapy general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed Neutralization assay for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their Supported lipid bilayer Label-free analysis administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical tech­ nique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 102 TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentra­ tions (IC50) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer. 1. Introduction made for the development and distribution of rapid detection tests (Dinnes et al., 2021; Singh et al., 2022; Thapa et al., 2022; Wang et al., Since December 2019, research in coronavirus infectious disease 2020) and vaccines (Creech et al., 2021) that so far have successfully (COVID-19) has become a hot topic in the field of sensors and analytical allowed an efficient population screening, slowing down viral trans­ techniques for diagnostics as well as in vaccine and therapy develop­ mission, and greatly reducing clinical complications and deaths by ment. COVID-19, caused by the SARS-CoV-2 (Severe Acute Respiratory SARS-CoV-2 infection. However, the administration of antiviral thera­ Syndrome Coronavirus 2), was declared a pandemic by the World Health pies for an early treatment of the disease seems to have found significant Organization (WHO) in early 2020 (Hadi et al., 2020; Mohan and Vinod, difficulties, becoming a bottleneck in the clinical assistance. 2020; Sheervalilou et al., 2020), with a rapid worldwide spread that Early antiviral therapies for COVID-19 are a critical need in high-risk accumulates hundreds of million cases and millions of deaths (Sen-­ individuals with comorbidities, weaken immune systems, or limited Crowe et al., 2020). Furthermore, the COVID-19 pandemic has seriously access to vaccination (AminJafari and Ghasemi, 2020; Wouters et al., challenged many healthcare systems with a drastic work overload and a 2021). Besides of the desired effectiveness to prevent and control lack of resources to provide rapid diagnostic and prognostic informa­ SARS-CoV-2 infection with minimum side effects, they should be widely tion, and novel treatments for effective patient management (Kevadiya available, affordable, and suitable for non-hospitalized patients. Among et al., 2021; Ludwig and Zarbock, 2020). Significant efforts have been different candidates, such as the convalescent plasma (Li et al., 2020) or * Corresponding author. ** Corresponding author. E-mail addresses: maria.soler@icn2.cat (M. Soler), laura.lechuga@icn2.cat (L.M. Lechuga). https://doi.org/10.1016/j.bios.2023.115137 Received 14 October 2022; Received in revised form 16 January 2023; Accepted 6 February 2023 Available online 8 February 2023 0956-5663/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 the antivirals paxlovid and remdesivir (AminJafari and Ghasemi, 2020; their ability to block and neutralize virus infectivity will be measured by Pashaei and Rezaei, 2020), the most promising ones have been the either counting the number of formed plaques or the inhibition of the monoclonal antibody (mAb) immunotherapies (Pashaei and Rezaei, cytopathic effect (i.e., microneutralization test), resulting in a titration 2020). Monoclonal antibodies are recombinant or in vitro produced curve from which the optimum dose and the half maximum inhibitory immunoglobulin proteins that mimic the immune system’s ability to concentration (IC50) are determined. The main drawbacks of this tech­ prevent and stop the coronavirus infection by binding to external viral nique are the long times required for cell culture and plaque forming by spikes (S protein), inhibiting its interaction with the host cell receptors, viral infection that can extend from several hours to days. Also, the and therefore neutralizing virus entry and replication in the human complexity, specialization, and laborious procedures for microbiology organism (Fig. 1) (AminJafari and Ghasemi, 2020). A few mAb therapies and cell culturing inevitably increase the costs and price of the therapy have been approved by the American Food and Drug Administration (Iacob and Iacob, 2020). (FDA) and the European Medicine Agency (EMA) between 2021 and To overcome these limitations, we introduce a new biomimetic label- 2022 for the treatment of mild to moderate COVID-19 patients, like free plasmonic sensor for simplifying and accelerating the screening and bamlanimivab, regdanvimab, sotrovimab, or the cocktails casir­ evaluation of novel mAb immunotherapies for COVID-19. This novel ivimab/imdevimab and tixagevimab/cilgavimab (Drożdżal et al., 2020; sensing technology provides accurate, reliable, and precise neutraliza­ Plaze et al., 2021). Nonetheless, their late approval and high prices tion information with a minimum sample and reagent consumption, evidence the challenging, laborious, and time-consuming procedures rapid assays, and user-friendly operation. In particular, we propose the required to develop and evaluate antiviral mAb therapies, which could use of a Surface Plasmon Resonance (SPR) biosensor as a mature tech­ became a major limitation for their general implementation in health nology, worldwide commercially available, automatable, and with systems (Niknam et al., 2022; Sparrow et al., 2017). The selection and demonstrated reliability for pharmacology studies, diagnostics, and validation of mAb immunotherapies is generally carried out via therapy evaluation applications (Nguyen et al., 2015; Soler and Huertas, neutralization assays (Payne, 2017). Initial steps of the protocol include 2022). SPR biosensor systems are based on the evanescent field sensing high-throughput screening techniques to identify antibodies with high principle that enables the label-free monitoring of biochemical and affinity toward the S protein (or its receptor binding domain, RBD), biomolecular interactions in real time with excellent sensitivities often performed with multiplexed bead-based assays (e.g., Luminex) or (pM-nM range). Most common SPR sensors employ a nanometer-thin enzyme-linked immunosorbent assays (ELISA), among others. Once layer of gold (45–50 nm) as transducer, in which the propagating elec­ selected, antibodies are evaluated through an infectivity or neutraliza­ tromagnetic resonance (i.e., collective oscillation of conduction band tion assay. This assay consists in incubating the antibody at different electrons) is excited through prism-coupled illumination with a polar­ titers with purified samples of the virus – usually employing a pseudo­ ized light beam (Kretschmann configuration) (Soler and Lechuga, 2021). typed virus (Lei et al., 2020); and these samples are added to a dish of This plasmonic resonance generates the electromagnetic field that cultured cells representing the host. In the absence of neutralizing an­ evanescently penetrates into the adjacent dielectric up to a few hundreds tibodies, the viruses will infect the host cells generating a number of of nanometers (100–300 nm), probing the biomolecular interactions as a plaques and areas lacking cells. At increasing antibody concentrations, response to the variation of the refractive index of the medium. The Fig. 1. Schematic illustration of the biomimetic plasmonic biosensor approach for mAb therapy evaluation via neutralization assays. 1) panel shows direct capture and detection of SARS-CoV-2 viruses over an artificial cell membrane displaying ACE-2 receptors created on the plasmonic sensor. 2) panel shows how therapeutic monoclonal antibodies bind to the Spike protein of the SARS-CoV-2 virus, blocking and inhibiting its interaction with the host cell. 2 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 interrogation of certain properties of the light, such as angle, intensity or phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L- wavelength, provides quantitative information about the concentration serine) were provided by Avanti Polar Lipids (Alabaster, Alabama, USA). of the analyte captured on the sensor surface and also the affinity and Reagents for buffer preparation (10 mM PBS (Phosphate buffer saline, kinetic parameters of the biomolecular interaction. In the last two years, pH 7.4), 50 mM HEPES (N-(2-Hydroxyethyl) piperazine-N-(2- plasmonic biosensors have been demonstrated for different applications ethanesulfonic acid, pH 7.24), and MES (2-(N-morpholino) ethane sul­ in COVID-19, primarily for antigen- and antibody-targeted diagnostics, fonic acid, pH 5.0)) and reagents for carboxylic groups activation (1- but also for the study of the biomolecular interactions involved in the ethyl-3(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) SARS-CoV-2 infection cycle (Behrouzi and Lin, 2022; Hassan et al., and sulfo-N-hydroxysuccinimide (s-NHS)) were purchased to Sigma- 2021; Mauriz and Lechuga, 2021; Shrivastav et al., 2021; Syed Nor et al., Aldrich (Steinhem, Germany). Angiotensin-converting enzyme 2 (ACE- 2022; Szunerits et al., 2022; Yano et al., 2022; Ye et al., 2021; Saad et al., 2), SARS-CoV-2 S proteins, and neutralizing monoclonal antibody 2 2022), with detection limits in the 1–100 ng/mL range for proteomic (NAb2) were acquired from GenScript (Leiden, The Netherlands). The assays and 103–105 viruses/mL for cell and genomic analysis, and for the neutralizing monoclonal antibody 1 (Nab1) was acquired from Sino screening and profiling of therapy candidates, including antibodies Biologicals (Eschborn, Germany). The inactivated SARS-CoV-2 viruses (AminJafari and Ghasemi, 2020). However, these publications only Omicron BA.1 (hCoV-19/Italy/LAZ-INMI-2890/2021, GISAID accession address isolated biomolecular interactions, employing in-solution re­ ID EPI_ISL_7716384) and B1.617.2/delta (hCoV-19/Italy/LAZ-INMI- combinant proteins with three-dimensional (3D) freedom that do not 648/2021 GISAID accession ID EPI_ISL_2000624) were kindly provided take into account the intrinsic cell physiology and microenvironment by our collaborator Dr. Giulia Matusali from The National Institute of restrictions in terms of mobility and number of interactions (Soler et al., Infectious Diseases Lazzaro Spallanzani (INMI, Rome, Italy). The virus 2018). production and inactivation procedures have been described in (Ruiz-­ The main novelty and innovation of our work reside on a two- Vega et al., 2022). dimensional (2D) biomimetic assay approach employed to increase the reliability and transferability of the therapy screening tests while 2.2. SPR biosensor description reducing the analysis times and the overall complexity of the evaluation study. Our biosensor is functionalized with an artificial cell membrane Our proprietary SPR biosensor based on Kretschmann configuration displaying the host cell receptors utilized by the SARS-CoV-2 for entry is a self-designed and assembled compact and small device able to and infection, i.e., the angiotensin-converting enzyme 2 (ACE2), and the monitor binding events in real-time through resonance wavelength neutralization assay is performed with ultraviolet (UV)-inactivated interrogation at a fixed angle of incidence (θ = 70o). The plasmonic SARS-CoV-2 viruses, which maintain the structural properties and pro­ sensor chip is sandwiched in between a trapezoidal glass prism linked tein activity and can be safely manipulated in P2-level biosafety labo­ through a refractive index matching oil (n = 1.512) and a customized ratory installations (Ruiz-Vega et al., 2022). With this methodology, we polymeric single-channel flow cell. The flow cell is connected to a fluidic can more accurately simulate the virus-cell interaction during an delivery system composed of a syringe pump and a hand-functioned infectivity assay – where both target molecules are anchored to their injection valve including a loop (150 μL) for sample injection. The sy­ respective cell membranes - while eliminating the need of host cell ringe pump delivers a constant volume flow at a fixed rate that can be culture procedures. Artificial cell membranes formed by planar lipid adjusted. A collimated TM-polarized broadband light source is used to bilayers have been previously demonstrated and applied in biosensing excite the sensor surface through prism coupling and the reflected light for cell biology studies (e.g., protein-membrane interactions (Dahlin is collected and fiber-coupled to a CCD mini-spectrometer (Jaz Module, et al., 2005), for drug discovery research (Limaj et al., 2016), and also Ocean Optics, Orlando, FL, USA). The real-time monitoring of shifts in for the evaluation of cell immunotherapies for cancer (Soler et al., plasmonic resonance peak (λSPR) can be read out by polynomial fitting 2018). We take advantage of this unique biomimetic scaffold to attain using a user-friendly home-made software developed in open-source and deliver for the first time an innovative, convenient, and highly programming scripts (Python). Au plasmonic thin films (1 nm Ti, 49 versatile biosensor for efficient screening and evaluation of mAbs as nm Au) were coated with a thin layer of SiO2 (10 nm) to provide a antiviral immunotherapies for COVID-19. To demonstrate the capabil­ hydrophilic surface for efficient lipid bilayer formation. Before ities of our innovative technology, we analyze the interaction of inac­ mounting the plasmonic chip on the SPR sensor, a specific protocol for tivated SARS-CoV-2 viruses of different variants with an chip cleaning was followed. Firstly, the chip was sonicated 1 min at ACE2-functionalized membrane and the neutralizing effect of different room temperature in various solvents acetone, ethanol, and MiliQ water, dosage of monoclonal antibodies. Our biomimetic sensor enables a rapid respectively, then dry with an N2 stream. To make the sensor surface comparison of different antibody candidates as well as different virus more hydrophilic, the plasmonic chip was placed in UV/Ozone variants, and it can provide accurate data on the optimum inhibition Procleaner Plus (Bioforce Nanosciences, Utah, US) for 30 min. Optical concentration (IC50). The new methodology demonstrated here could characterization of the SPR biosensor is detailed in the Supporting In­ position biosensor technologies beyond the state of the art – mainly formation (Fig. S1). focusing in diagnostic detection analysis and biomolecular interaction characterization – by providing a versatile and powerful device to be 2.3. Supported lipid bilayer (SLB) formation and ACE-2 receptor employed for the evaluation of novel biomolecular therapy formulations immobilization (e.g., nanobodies), for diagnostic seroneutralization assays, for vaccine development, etc. We foresee the implementation of plasmonic bio­ The functional supported lipids bilayer (SLB) was formed by sensors in biomedical and pharmaceutical laboratories as a valuable tool disruption of small unilamellar vesicles (SUV) over a hydrophilic SiO2 to accelerate, simplify, and reduce costs in the development of more surface. A specific volume of different lipid formulations (POPC:DOPS, effective, personalized, and universal therapies that can boost health­ 10:1) was dissolved in chloroform and mixed in a glass vial to obtain 1 care around the world. mg of dry lipids. To evaporate the solvent residues and form a dry lipids thin film, the vial was stored in a vacuum desiccator overnight in 2. Experimental section continuous N2 flux at room temperature. To rehydrate the lipid’s thin film, 1 mL PBS buffer (10 mM, pH 7.4) was added up to get 1 mg/mL 2.1. Chemical and biological reagents concentration. The final solution was vortexed for 2–3 min and soni­ cated at room temperature for 1 h to make a heterogenous vesicles so­ Acetone and ethanol were purchased from Panreac Applichem lution. The vesicles were extruded into small unilamellar vesicles (SUVs) (Barcelona, Spain). Lipids (POPC, 1-palmitoyl-2-oleoyl-snglycero-3- through a polycarbonate membrane pore size (0.1 μm) with 100 3 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 extrusion cycles at 60o 0C temperature by using a mini-extruder (Avanti molecules (Peláez et al., 2020, 2018; Soler et al., 2016a) for different Polar Lipids, Inc., Alabaster, AL, USA). The final SUV solution was applications, generally employing a functional chemical matrix based on quickly injected through the sensor surface to create a lipids bilayer on alkanethiol self-assembled monolayers (SAMs) for direct gold sensor SiO2-coated Au thin film. After, a cleaning step of 10 mM NaOH was surface functionalization. In this work, the formation of artificial cell carried out to ensure a stable formation of a planar lipid layer. Once the membranes based on supported lipids bilayer (SLB) replaces the rigid functional SLB layer was stabilized, angiotensin-converting enzyme 2 SAMs, providing a 2D biomimetic scaffold that allows a slight bio­ receptors fused to a human Fc recombinant molecule (ACE-2, 50 μg/mL) receptor lateral mobility to form clusters and enhancing cell-cell in­ were covalently anchored through EDC/NHS (0.4 M/0.1 M) chemistry teractions (Soler et al., 2018). onto the functional SLB layer in MES buffer (50 mM, pH 5). To deacti­ Planar and functional SLBs can be formed onto the sensor surfaces vate the unreacted COOH groups, ethanolamine (EA, 1 M, pH 8) was through a well-established methodology based on spontaneous disrup­ injected for 1 min. All the measurements were carried out with PBS as tion of small unilamellar vesicles (SUV) upon contact with hydrophilic running buffer at 20 μL/min flow rate. surfaces, like SiO2 (Hardy et al., 2013; Kilic and Kok, 2016; Neupane et al., 2018; Richter and Brisson, 2005). In this sense, we have included a 2.4. SARS-CoV-2 virus detection and neutralization assays 10 nm SiO2 coating on the gold sensors, which will ensure the long-term hydrophilicity of the substrate and the stability of the lipid layer The optimization and evaluation of the virus detection assay were (Fig. 2a) (Ulmefors et al., 2021). The large variety of existing lipid for­ performed with the biomimetic ACE-2 functionalized sensor chip mulations allows preparing vesicles with different mixtures that incor­ employing two variants of SARS-CoV-2: Delta and Omicron (using the porate desired functionalities (e.g., amine, carboxyl, biotin, etc.) for viral antigens and whole UV-inactivated SARS-CoV-2 virus). The cali­ subsequent conjugation with the cell receptor. We have selected a bration curves for both viral antigens and whole viruses were generated mixture of phosphocholine (1-palmitoyl-2-oleoyl-snglycer­ by measuring concentrations ranging from 30 ng/mL to 5000 ng/mL o-3-phosphocholine, POPC) at 90% molar ratio with a carboxyl (-COOH) and 103 TCID50/mL to 5 × 105 TCID50/mL, respectively, in standard functional phosphoserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, buffer (HEPES, 50 mM, pH 7.4) at 20 μL/min flow rate. After each DOPS) at 10% molar ratio. SUVs were prepared by extrusion, employing sample, the receptor-analyte interactions were disrupted and the bio­ 0.1 μm size filtration membranes, resulting in an homogeneous sus­ surface was regenerated by injecting 10 mM NaOH solution for 2 min at pension of approximately 100 nm vesicles in PBS buffer. The SUV sus­ the same flow rate. For neutralization assays, virus samples containing pension was readily injected on the SPR biosensor and the formation of the mAbs at different concentrations (from 0 to 5000 ng/mL) were pre- the SLB onto the sensor surface was monitored in real time (Fig. 2b). The incubated for 10 min at room temperature and then flowed over the sharp increase of the sensor response (corresponding to a cooperative biofunctionalized surface. Data were analyzed using Origin 8.0 (Ori­ binding equation), reached equilibrium at Δλ = 14 nm, indicating that ginLab, MA, US) and GraphPad Prism 8 (GraphPad Software, CA, US). SUVs instantly disrupted over the sensor surface and promptly formed a Detection calibration curves were plotted as the mean and standard uniform SLB layer. A cleaning solution (NaOH 10 mM) was flowed over deviation (mean ± SD) of the acquired biosensor response (Δλ) versus the bilayer for 2 min to confirm stability (i.e., nothing was removed from the target concentration. The data were fitted to a two-site specific the surface). After, we covalently immobilized ACE-2 receptors (50 binding model curve. The limit of detection (LOD), defined as the μg/mL) on the COOH-functional SLB by means of the well-known smallest concentration distinguishable from the blank, and the limit of EDC/NHS chemistry (Fig. 2c). We employed a recombinant human quantification (LOQ), defined as the minimum concentration that can be ACE-2 – Fc chimera in order to facilitate its anchoring onto the lipid reliably detected and quantified, were determined as the concentration surface and ensure accessibility of the viruses. The whole bio­ corresponding to 3 and 10 times the standard deviation of the baseline of functionalization procedure was carried out in situ at room temperature the sensor signal, respectively. Inhibition calibration curves were and was completed within approximately 2 h. To confirm the robustness plotted as the mean and standard deviation of the biosensor response and reproducibility of the procedure for artificial cell membrane for­ normalized to percentages. The data were fitted to a dose-response in­ mation, we determined the coefficient of variability for both SLB for­ hibition fitting curve. The half maximal inhibitory concentration (IC50) mation and ACE-2 immobilization, obtaining values of 7% and 14%, can be defined as the 50% quantity of drug required to inhibit a bio­ respectively, over more than 25 biofunctionalization processes. logical process. This value can be calculated by testing different analyte concentrations in triplicates and signals (mean ± SD) plotted vs the 3.2. Analysis of SARS-CoV-2 virus interaction with ACE-2 cell receptors logarithmic value of the analyte concentration. All experiments for the virus detection were carried out in the ICN2 BSL2 facilities with The artificial cell membrane scaffold prepared on the plasmonic approval of the Biosafety Committee of the Autonomous University of sensor surface was first used to study and evaluate the binding inter­ Barcelona (UAB) (HR-599-20). action between the ACE-2 receptors and the SARS-CoV-2 viruses of both variants. Initially, we employed recombinant viral antigens corre­ 3. Results and discussion sponding to the Spike (S) protein of Delta and Omicron SARS-CoV-2 to confirm specific detection of the viruses and to assess possible affinity 3.1. Formation of artificial cell membranes expressing ACE-2 receptors differences between the two variants (Fig. 3a and Fig. S2). The cali­ bration curves for SARS-CoV-2 viral antigens over a wide range of We have employed a custom-designed proprietary SPR biosensor concentrations (15–5000 ng/mL) were plotted against the biosensor based on prism-coupling excitation and wavelength interrogation for the response (Δλ); the limits of detection (LOD) and limits of quantification creation of an artificial host cell interface displaying ACE-2 receptors, (LOQ) were determined at 18 ng/mL and 49 ng/mL, respectively, for the which provides a nature-imitating scaffold for therapeutic mAb evalu­ Delta variant, and at 12 ng/mL and 37 ng/mL, respectively, for the ation through SARS-CoV-2 neutralization assays. This biosensor pro­ Omicron variant. These excellent sensitivity values prove the powerful vides label-free and real-time monitoring of biological interactions with performance of the SPR biosensor for highly accurate biomolecular excellent sensitivity in a user-friendly environment and operation. Our detection assays, and indicate no major affinity differences of the ACE-2 biosensor technology has previously proved itself as a potential candi­ receptor towards the two virus variants. date for clinical diagnostics and biomedical research, including direct Since our main goal is to develop a close-to-nature biomimetic assay, detection of proteins (Aviñó et al., 2019; Calvo-Lozano et al., 2022; we also employed inactivated SARS-CoV-2 viruses as main target. The Pelaez et al., 2020; Soler et al., 2016b, 2015, 2014), nucleic acids SARS-CoV-2 viruses were produced and provided by the National (Calvo-Lozano et al., 2020; Huertas et al., 2018, 2016), and small Institute of Infectious Diseases (INMI) of Rome (Italy), and they were 4 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 Fig. 3. Standard calibration curve for a) SARS-CoV-2 viral antigens (Delta and Omicron) obtained with triplicate measurements of different concentrations over a range between 30 ng/mL to 5000 ng/mL for each specific protein variant; and b) UV-inactivated SARS-CoV-2 virus different variants (delta and omicron) obtained with triplicate measurements of different virus titters over a range between 103 TCID50/mL to 5 × 105 TCID50/mL for each specific virus variant. duly inactivated through ultraviolet (UV) irradiation (15 min, λ 254 nm), which was verified by inoculating the viruses on VeroE6 cells and confirming no viral replication for three consecutive passages. The UV inactivation treatment is intended to only affect the genomic material of the virus (RNA), therefore unaltering the external structure of the viral particles allowing their safely use in BSL2 laboratories (Ruiz-Vega et al., 2022). SARS-CoV-2 samples at different titers (103 to 5 × 105 TCID50/mL) were prepared by serial dilution in standard buffer (HEPES 50 mM pH 7.4) and flowed over the biomimetic scaffold on the sensor surface. We obtained calibration curves for the Delta and Omicron variants, showing a proportional increase of the sensor signal with the viral load (Fig. 3b and Fig. S2). The LODs were determined at 630 Fig. 2. (a) Schematic illustration of the ACE-2 functional supported lipid TCID50/mL and 200 TCID50/mL for Delta and Omicron variants, bilayer formed on SiO2-coated plasmonic sensor surface; (b) SPR sensorgram respectively. The LOQs were determined at 4700 TCID50/mL and 1200 showing the formation of a lipid bilayer from the disruption of small uni­ TCID50/mL for Delta and Omicron variants, respectively. Here, we lamellar vesicles (SUV), followed by a NaOH cleaning step; (c) SPR sensorgram showing the ACE-2 receptor immobilization on the COOH-functional SLB. perceived that the binding of Omicron SARS-CoV-2 viruses to the ACE-2 receptor is slightly better (25% higher binding efficacy) than the Delta SARS-CoV-2 virus. The reason could be attributed to the spontaneous 5 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 mutations occurred within the Omicron S protein, presenting a higher affinity towards the cell receptors, becoming more contagious, as it has been observed during the evolution of the COVID-19 pandemic. None­ theless, it could also be due to unavoidable experimental variations in the production, inactivation, or manipulation of the viral samples during the study. The sensitivity values obtained for the direct and label-free detection of SARS-CoV-2 viruses with our novel biomimetic sensor are outstanding compared to the state-of-the-art. To our knowledge, only a recent study has reported a plasmonic sensor for the quantification of whole SARS-CoV-2 virus particles, achieving a detection limit of 370 viral particles per mL (vp/mL) (Huang et al., 2021), which is within the same range of our approach but using pseudotyped viruses, a secondary amplification step with gold nanoparticles, and not validated with real samples. On the other hand, our group developed a nanophotonic interferometric sensor for COVID-19 diagnostics demonstrating direct SARS-CoV-2 virus detection with detection limits below 100 TCID50/mL (Ruiz-Vega et al., 2022). Herein, we have achieved similar results employing a conventional and user-friendly SPR technology with an innovative sensor biofunctionalization, which is considered to enhance the SARS-CoV-2 virus binding interaction by mimicking the human cell micro-environment. In fact, different experiments performed for the detection of SARS-CoV-2 viruses using conventional bio­ functionalization with self-assembled monolayers resulted in lower sensitivity and worse reproducibility (see Supporting Information, Fig. S3). Furthermore, it is important to remark that the range of virus concentrations employed in this study correspond to most common viral loads found in COVID-19 patients at early stages of the infection (Caplan et al., 2021; Soria et al., 2021), being therefore appropriate for the evaluation of antiviral therapies for early treatment. 3.3. Evaluation of binding affinity and neutralization capacity of different monoclonal antibodies with SARS-CoV-2 viral antigens For the development of the biosensor-based neutralization assay, we selected two different commercial SARS-CoV-2 neutralizing mAbs (NAb): NAb1, which is a recombinant IgG produced towards the wild- type SARS-CoV-2 Spike protein (S1 fraction) and the NAb2, which is a recombinant IgM produced against a recombinant SARS-CoV-2 RBD protein. A preliminary evaluation study was carried out to test and confirm the reactivity and affinity of both NAb towards the two variants Fig. 4. a) binding curves for Delta-S protein with NAb1 and NAb2, respec­ of the SARS-CoV-2 virus. To that, we prepared a SPR sensor chip func­ tively; and b) Standard binding curves for Omicron-S protein with NAb1 and tionalized with each viral antigen (Delta-S protein and Omicron-S pro­ NAb2, respectively. tein), immobilized onto a conventional alkanethiol SAM through EDC/ NHS chemistry. Various concentrations of NAb1 and NAb2, in the range of 15 ng/mL to 200 ng/mL, were flowed over each immobilized viral reduce and eventually impede the interaction of the viral antigens with antigen to obtain concentration-dependent binding curves (Fig. 4a and the immobilized ACE-2 receptor, achieving complete blocking of the b). We also tested a negative control sample (SARS-CoV-2 Nucleocapside interaction at concentrations higher than 2000 ng/mL. Slight differences (N) protein at 200 ng/mL) that resulted in negligible non-specific could be readily observed when comparing the two different NAbs, as interaction with each viral antigen. These binding curves demonstrate the assays with NAb2 (neutralizing IgM) resulted in an early reduction of an appropriate affinity of both neutralizing antibodies towards each the signal compared to the assays with NAb1 (neutralizing IgG). These viral antigens, and therefore their capability to interfere in the interac­ differences were corroborated with the determination of the half tion between the virus and the cell receptor to prevent infection (Xiaojie maximum inhibition concentration (IC50); for the Delta variant assay, et al., 2021). the IC50 (NAb1) was 142 ng/mL, while the IC50 (Nab2) was 68 ng/mL, Once the affinity and binding interaction of the neutralizing anti­ and for the Omicron variant assay, the IC50 (Nab1) was 122 ng/mL and bodies with the target viral antigens was confirmed, we proceeded to the IC50 (Nab2) was 39 ng/mL. These results could be explained by perform competitive immunoassays with the same recombinant viral intrinsic structural differences between both Nabs. An IgG is a Y-shaped antigens in order to test and evaluate the effectiveness of the antibodies bivalent molecule with 2 antigen binding sites at their short arms, while to inhibit the interaction with the cell receptor immobilized on the an IgM is a pentamer biomolecule with up to 10 antigen binding sites. It artificial cell membrane (Fig. 5a). Different dilutions of NAb1 and NAb2 can be therefore assumed that IgMs would be more efficient to trap viral (7.8–5000 ng/mL) were incubated for 10 min with Delta S-protein and antigens in solution, and therefore able to accelerate the blocking of Omicron S-protein, respectively, at a fixed concentration (500 ng/mL) their interaction with ACE-2 receptors. (Fig. 5b and c). Representative sensorgrams are shown in Supporting Information (Fig. S4). We also included a blank sample (viral antigen 3.4. Demonstration and validation of the biomimetic SPR neutralization sample with no NAb), and a negative control sample (NAb at 5000 ng/ assays for SARS-CoV-2 viruses with different neutralizing antibodies mL with no viral antigen), which resulted in null or negligible signal. As expected, in all cases, the presence of neutralizing antibodies was able to Finally, we addressed the validation of our biomimetic plasmonic 6 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 Fig. 5. Competitive immunoassay with SARS-CoV-2 viral antigens and virus variants: a) Schematic illustration of the neutralization assay performed with SARS-CoV- 2 viral antigens and neutralizing antibodies (NAb) over an ACE-2 receptor anchored to a SLB-functionalized SPR biosensor; b) Inhibition curves obtained for the Delta-S protein at a fixed concentration (500 ng/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2); c) Inhibition curves obtained for the Omicron-S protein at a fixed concentration (500 ng/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2). All sensor signals are normalized according to maximum signal (100%) and minimum signal (0%) to facilitate comparison; d) Schematic illustration of the neutralization assay performed with SARS-CoV-2 viruses and neutralizing antibodies (NAb) over an ACE-2 receptor anchored to a SLB-functionalized SPR biosensor; e) Inhibition curves obtained for the Delta SARS-CoV-2 at a fixed concentration (5 × 104 TCID50/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2); f) Inhibition curves obtained for the Omicron SARS-CoV-2 viruses at a fixed concentration (5 × 104 TCID50/mL) incubated with different concentrations of both neutralizing antibodies (NAb1 and NAb2). All sensor signals are normalized according to maximum signal (100%) and minimum signal (0%) to facilitate comparison. sensor for the evaluation of neutralizing mAbs with the SARS-CoV-2 assay performed with viral antigens in solution, which reveals the viruses. The virus competitive immunoassays were carried out simi­ importance of carrying out the assays in a biomimetic manner, with both larly to the ones performed with viral antigens. A fixed concentration of ACE-2 receptor and Spike proteins embedded in their corresponding cell UV-inactivated SARS-CoV-2 virus sample (5 × 104 TCID50/mL) was membranes. Despite the fact that NAb2 is an IgM molecule with more incubated with different concentrations of NAb1 and NAb2 (7.8–5000 binding sites available, the steric hindrance limits its capability to ng/mL) for 10 min, and then directly introduced in the sensor func­ interact with a large number of antigens and the inhibition capability tionalized with an artificial cell membrane displaying ACE-2 receptors results similar to the one of the IgG antibodies. On the other hand, the (Fig. 5d). Dose-response inhibition curves were obtained for each SARS- superior capability of both antibodies for neutralizing the Omicron CoV-2 variant (Delta and Omicron) and for each neutralizing antibody variant more efficiently than the Delta variant is maintained, with (Fig. 5e and f). Representative sensorgrams are shown in Supporting overall lower IC50 values and achieving a complete inhibition of the Information (Fig. S5). The IC50 values for each assay were calculated at interaction with a lower dose (approximately 250 ng/mL). 100 ng/mL and 78 ng/mL for the Delta variant with NAb1 and NAb2, The comparison of this study with those performed for some of the respectively; and 44 ng/mL and 22 ng/mL for the Omicron variant with approved mAb immunotherapies, such as casirivimab, imdevimab, NAb1 and NAb2, respectively (Table 1). Herein, the differences between bamlanimab and sotrovimab, shows that the IC50 values determined in the two NAbs are not significant, compared to the 3D neutralization our biomimetic sensing assay are in good agreement with the values determined through standard techniques (ELISA), which range between Table 1 10 and 1000 ng/mL (Hwang et al., 2022), confirming the accuracy of the Summary of main analytical parameters. analysis. Furthermore, it is important to remark that the biomimetic SPR sensor offers key advantages over the conventional techniques, like the SARS-CoV-2 target Direct & Label-free Detection Neutralization Assay and variant rapidness and possibility to automate the assays - with no need of LOD LOQ IC50 IC50 amplification, washing, or labelling processes -, the reliability of per­ (NAb1) (NAb2) forming close-to-nature analysis without the complex and Delta S protein 18 ng/mL 49 ng/mL 142 ng/ 68 ng/mL time-consuming mammalian cell culturing, and employing a small vol­ mL ume of virus sample (150 μL) at concentrations analogue to common Omicron S protein 12 ng/mL 36 ng/mL 122 ng/ 39 ng/mL mL viral loads found in patients with mild or moderate symptomatology. All Delta SARS-CoV-2 630 4.8 × 103 100 ng/ 78 ng/mL these characteristics become critical when addressing the evaluation of virus TCID50/mL TCI50/mL mL novel therapeutic candidates, and especially in a global health emer­ Omicron SARS-CoV- 200 TCI50/ 1.2 × 103 44 ng/mL 22 ng/mL gency like the COVID-19 pandemic. The possibility to perform simpler, 2 virus mL TCI50/mL reagent-less, faster, and more reliable therapy screening and assessment 7 R. Batool et al. Biosensors and Bioelectronics 226 (2023) 115137 studies could readily accelerate the selection and regulation of the most project (Ref. 101003544), funded by the H2020 Research and Innova­ promising formulations, reducing the production and manufacturing tion Programme of the European Commission (H2020-SC1-PHE-Coro­ costs, and boosting their implementation in healthcare systems navirus-2020). RB acknowledges the economic support (Ref. PRE2019- worldwide. 089357) from MCIN and AEI linked to the project SEV-2017-0706-19-4. MS acknowledges the economic support of the Ramon y Cajal Grant 4. Conclusions (RYC2020-029015-I) funded by the AEI/MCIN and the European Union NextGenerationEU/PRTR. INMI also acknowledges funding from the We have developed a rapid, simple and user-friendly biomimetic European Virus Archive – GLOBAL (Grant No. 871029). The ICN2 is sensing assay to reduce the overall complexity of SARS-CoV-2 neutral­ supported by the CERCA programme (Generalitat de Catalunya) and the ization studies and to accelerate the production and optimization of new Severo Ochoa Centres of Excellence programme, funded by the AEI COVID-19 immunotherapies based on monoclonal antibodies. Our (Grant. No. SEV-2017-0706). plasmonic sensor is functionalized with an artificial cell membrane that imitates the real virus-cell interactions, enabling label-free monitoring Appendix A. Supplementary data of the biomolecular infection mechanism (SARS-CoV-2 S protein binding to ACE-2 receptor) in real time, without introducing external tags, and Supplementary data to this article can be found online at https://doi. reaching an excellent sensitivity, with detection limits in the 102 org/10.1016/j.bios.2023.115137. TCID50/mL range for two major variants of concern of SARS-CoV-2 viruses (Delta and Omicron), which is one order of magnitude better References than the majority of plasmonic biosensors reported so far (103–104 vi­ ruses/mL). The biomimetic sensor has been applied for the evaluation of AminJafari, A., Ghasemi, S., 2020. The possible of immunotherapy for COVID-19: a systematic review. Int. Immunopharm. 83, 106455 https://doi.org/10.1016/j. two different monoclonal antibodies as potential candidates for early intimp.2020.106455. COVID-19 treatment. 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Maria Dinnes, J., Deeks, J.J., Berhane, S., Taylor, M., Adriano, A., Davenport, C., Dittrich, S., Soler conceived and designed the project, supervised the experiments Emperador, D., Takwoingi, Y., Cunningham, J., Beese, S., Domen, J., Dretzke, J., and carried out data analysis and discussion. RB and MS prepared the Ferrante di Ruffano, L., Harris, I.M., Price, M.J., Taylor-Phillips, S., Hooft, L., Leeflang, M.M.G., McInnes, M.D.F., Spijker, R., Van den Bruel, A., Arevalo- manuscript. INMI produced and purified the UV-inactivated SARS-CoV- Rodriguez, I., Buitrago, D.C., Ciapponi, A., Mateos, M., Stuyf, T., Horn, S., 2 samples. LML coordinates the research and supervised experimental Salameh, J.P., McGrath, T.A., van der Pol, C.B., Frank, R.A., Prager, R., Hare, S.S., and discussion activities. All authors have read, revised, and approved Dennie, C., Jenniskens, K., Korevaar, D.A., Cohen, J.F., van de Wijgert, J., Damen, J. 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