Lab on a Chip This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online COMMUNICATION View Journal | View Issue Selection and characterisation of bioreceptors to Cite this: Lab Chip, 2022, 22, 2938 develop nanoparticle-based lateral-flow immunoassays in the context of the SARS-CoV-2 Open Access Article. Published on 29 July 2022. Downloaded on 11/2/2022 7:48:10 AM. Received 27th May 2022, Accepted 10th June 2022 outbreak† DOI: 10.1039/d2lc00486k Liming Hu, ‡a Enric Calucho,‡a Celia Fuentes-Chust,a Claudio Parolo, ab a Andrea Idili, Ruslan Álvarez-Diduk, a Lourdes Rivas a and Arben Merkoçi *ac rsc.li/loc This manuscript aims at raising the attention of the scientific the bioreceptors (e.g. antibodies or aptamers). However, the community to the need for better characterised bioreceptors for selection of suitable bioreceptors for the detection of SARS- fast development of point-of-care diagnostic devices able to CoV-2 unveiled several experimental hurdles, which we want support mass frequency testing. Particularly, we present the to share with the community. More specifically, we want to difficulties encountered in finding suitable antibodies for the raise attention towards the importance of comprehensive development of a lateral flow assay for detecting the characterisation of bioreceptors (in this case, antibodies) nucleoprotein of SARS-CoV-2. before their implementation into LFAs. Many studies compare the performance of commercial SARS-CoV-2 diagnostic Introduction kits,19–26 but to the best of our knowledge, only one recent work has focused on the technical challenges behind The COVID-19 pandemic1–3 has shown the importance of bioreceptor selection.27 developing reliable yet easy-to-use, cheap, fast, and portable The COVID-19 pandemic represents a unique situation diagnostic devices to support mass testing.4–10 Diagnostic due to the scarcity of antibodies against SARS-CoV-2 antigens testing is fundamental for a rapid screening of the at the beginning of the outbreak (from December 2019 to population, to identify and track positive cases (i.e. both May 2020). During this period, the research community made symptomatic and asymptomatic individuals), and immunity an important effort in the characterisation process to select assessment.11,12 As suggested by the World Health suitable antibodies to rapidly face the fast spread of the virus. Organization (WHO), in order to meet such a high demand of The main goal of this characterisation is to understand testing, countries have been relying on lateral flow assays whether the selected antibodies exhibit the required binding (LFAs).13 Indeed, such molecular sensing platforms allow properties to work in a LFA platform. Specifically, antibodies achieving the rapid (<30 min), low-cost (5 USD), and single- have to display: (1) stability, in order to work under variable step detection of the COVID-19 biomarkers.14 Moreover, LFAs environmental conditions (temperature, humidity, pressure) are an evolving platform with constantly improving and support long-term storage; (2) fast binding kinetics, due sensitivity.15–17 Our group recently provided a protocol to the short time window for the bioreceptor–analyte describing the fabrication of a LFA to detect human IgG.18 interaction in the LFA assay (in the range of seconds to a few This is a generalisable protocol that can be easily adapted to minutes); (3) strong binding affinity, as we want the signal to other targets, such as the SARS-CoV-2 virus, only by changing remain stable while and after the assay takes place.18 Unfortunately, besides already identified technical problems a Nanobioelectronics & Biosensors Group, Catalan Institute of Nanoscience and associated with antibodies such as batch to batch Nanotechnology (ICN2), CSIC, BIST, Campus UAB, 08193, Bellaterra, Barcelona, Spain. E-mail: arben.merkoci@icn2.cat differences,28–31 suppliers do not provide enough b ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital characterisation of important binding parameters (e.g. Clínic (Department of International Health), Universitat de Barcelona, Barcelona, binding and kinetic constants), and they test antibodies Spain using only standard laboratory procedures (e.g., ELISA, c ICREA, Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain western blot). A parallel approach could be the estimation of † Electronic supplementary information (ESI) available: Extended materials and methods, bioreceptor selection and characterisation data (PDF). See DOI: these parameters through thermodynamics, binding, and https://doi.org/10.1039/d2lc00486k kinetics studies, but this requires resources, time, and ‡ These authors contributed equally. facilities that private companies may not be willing to 2938 | Lab Chip, 2022, 22, 2938–2943 This journal is © The Royal Society of Chemistry 2022 View Article Online Lab on a Chip Communication implement.18 This lack of experimental data does not allow format, to check their compatibility with the conditions developers to pursue a rational selection of antibodies encountered in a LFA (i.e., under a constant flow in a forcing them into a trial-and-error approach. Since this can nitrocellulose membrane) (Fig. 2). Firstly, we performed ELISA hide many technical challenges, here we present a study tests to identify the antibody combinations with the best This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. which describes how to proceed in such circumstances with binding performance (Fig. 1A). In order to carry out these experimental tools that are more available and can be measurements, we followed a previously reported ELISA performed by any laboratory, always keeping in mind that protocol.34,35 The experimental criteria to identify such the final goal is the development of a colorimetric LFA to be antibodies were similar to the standard for evaluating antibody implemented in COVID-19 diagnostics, and considering that titre by the chessboard titration method, which allows similar scenarios can occur in the future. assessing two variables simultaneously: antibody couples and the presence/absence of nucleoprotein.36 Specifically, in the Open Access Article. Published on 29 July 2022. Downloaded on 11/2/2022 7:48:10 AM. Results and discussion absence of the target (i.e. blank), the background signal has to display a value lower than 0.2 a.u. (OD Blank), while in the Following our fabrication protocol for LFA,18 we characterised presence of a saturated concentration of nucleoprotein (100 ng antibodies for their adaptation as bioreceptors in an AuNP- mL−1), the produced signal should be higher than 1.0 a.u. (OD based LFA as they were released in the market. Specifically, Positive). Surprisingly, we found that out of 80 tested among the antigens of SARS-CoV-2, we decided to target the combinations, only 10 met these two criteria (Fig. 1B). Such a nucleoprotein since it is highly abundant in the virion.32,33 low success rate (12.5%) is a consequence of the high cross- From April 2020 to February 2021, we purchased 17 reactivity between antibodies (in most cases, a blank signal as commercial anti-nucleoprotein antibodies from different high as the positive can be observed (Fig. 1C)) and their low companies (see Table S1†). The criteria for the selection were: affinity for the target or slow kinetics (for antibody couples (1) cost, (2) antibody concentration (over 1 mg mL−1), (3) whose positive signals were lower than 1.0 a.u.) (Fig. 1D and E). delivery time, and (4) animal host used for its production. Since It should be noted that all but one antibodies were publicised the recognition event in LFAs is based on the formation of the to work in ELISA and that none of them were part of a pre- classic immune-sandwich complex (Fig. S3†), we screened validated matched pair. antibodies through two phases: (1) ELISA, to quickly check Among the 10 working antibody combinations, we antibody binding performance (Fig. 1) and (2) the half-stick selected the 5 combinations of capture and detection Fig. 1 ELISA tests were performed to screen antibody combinations with the best binding performance against the nucleoprotein (N protein) of SARS-CoV-2. (A) Schematic of the immune-sandwich ELISA procedure. (B) Antibody combinations with the best ELISA performance. The nucleoprotein concentrations of the blank and positive solutions are 0 ng mL−1 and 100 ng mL−1, respectively. Antibody combinations that meet the performance requirements should have the following conditions: the OD value of the blank nucleoprotein solution is less than 0.2 a.u. and the OD value of the positive nucleoprotein solution is greater than 1.0 a.u.27 (C–E) Each individual graph shows the ELISA test results of the same capture antibodies (coated on ELISA plate wells) combined with multiple detection antibodies. (C) Capture Abs H1 and detection Abs R1–R10 and M1/M5. None of the antibody combinations qualified because the OD value of the blanks was much greater than 0.2, which means high cross- reactivity and poor specificity; (D) capture Abs M1 and detection Abs R1–R1. Only M1/R6 and M1/R8 were qualified with OD blank <0.2 and OD positive >1.0 while the rest were not qualified due to OD blank >0.2 or OD positive <1.0; and (E) capture Abs M5 and detection Abs R1/R10. Couples M5/R1, M5/R6, M5/R7, M5/R8 and M5/R9 were satisfactory, especially M5/R1 and M5/R8, and the others were not qualified due to OD blank >0.2 (M5/R10) or OD positive <1.0. This journal is © The Royal Society of Chemistry 2022 Lab Chip, 2022, 22, 2938–2943 | 2939 View Article Online Communication Lab on a Chip This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 29 July 2022. Downloaded on 11/2/2022 7:48:10 AM. Fig. 2 Dot test for screening out antibody combinations and calibration curve of detection of nucleoprotein spiked in artificial saliva based on half-stick LFA. (A) Schematic representation of the dot test based on half-stick LFA: anti-nucleoprotein Abs (capture Abs) and secondary Abs were manually dropped on the nitrocellulose membrane as the test dot and control dot, respectively. When the half-stick is tested with a blank sample, only the control dot (or line) is visible, demonstrating that the assay functioned properly. If the sample contains nucleoproteins, both control and test dots (or lines) are visible, and the intensity of the latter will depend on the analyte concentration. (B) All antibody combinations with good performance in the dot test (easy to distinguish between blank and positive samples with the naked eye). R1/M5, R8/M4 and R8/M5 elicit the best response. (C) Calibration curve of the half-stick LFA with R8/M5 antibodies. The data were fitted to a four-parameter logistic curve (blue line). The fitted curve (obtained using Origin 2018 32-bit and presented as value ± standard error) corresponds to the following equation: y = start + (end − start) × x^n/(k^n + x^n), with start = 1.04 ± 1.09, end = 112.12 ± 10.21, k = 169.28 ± 44.06 and n = 0.90 ± 0.12. The reduced χ2 = 2.58, R2 = 1.00 and the adjusted R2 = 0.99 Grey points were beyond the curve range due to the saturation of bioreceptor binding sites. The dynamic range of the half-sticks is from 11.4 ng mL−1 to 791.0 ng mL−1, calculated from the signal change from 10% to 90%, and the graph was obtained by analysing at least three (n = 3) independent LFAs for each target concentration. The fitting curve, LoD, LoQ, and dynamic range fully were acquired by the protocol from Parolo et al.18 (D) Half-stick LFA for detection of nucleoprotein, responding to increasing concentrations of the nucleoprotein. antibodies (M2/R8, M5/R1, M5/R6, M5/R8 and M5/R9) with dot test results showed that three antibody couples (R1/M5, the best performance in order to move on to the half-stick R8/M4 and R8/M5) had similar test line peak values for the characterisation. To do this, we used dotted half-sticks rather detection of 100 ng mL−1 nucleoprotein (Fig. 2B). Then, than full LFA strips because they are faster to prepare (taking preliminary calibration curves of R1/M5, R8/M4 and R8/M5 into account the number of antibody combinations) and were obtained after testing the sensors with increasing smaller reagent amounts are required.18,37 In order to concentrations of nucleoprotein (0, 3, 10, 30, 100, 300, 1000, identify the antibodies with the best binding performance in and 3000 ng mL−1) (Fig. S5†). Fitting the curves with a four- the nitrocellulose membrane, we established that the blank's logistic parameter equation, we calculated the EC50, that is, signal should be <5.0 a.u. and the positive sample's signal the half maximal concentration of nucleoprotein that elicits a >30.0 a.u. These values approximately correspond with the response halfway between the baseline and saturation signal. dynamic range obtained in a provided protocol for human Given the immune-sandwich nature of the system, EC50 is a IgG detection.18 With these criteria, only 6 couples of good indicator of affinity. The R8/M5 couple displays the antibodies showed a suitable binding performance in the lowest EC50 value (134.7 ng mL−1), while the EC50 values for half-stick format (Fig. 2B). We hypothesise that the shorter R8/M4 and R1/M5 are 197.9 ng mL−1 and 337.1 ng mL−1 time of incubation/recognition and the absence of washing respectively (Fig. S5 and Table S4†). Moreover, R8/M5 showed steps in half-stick dot tests compared to ELISAs are the the best sensitivity for nucleoprotein detection, as observed causes of the decrease in the number of antibody couples from the steeper slope in the calibration curves (Fig. S5A and compatible with a paper-based platform. B†). Consequently, antibody couple R8/M5 was selected for To demonstrate the feasibility of the half-stick assay for the further development of a nucleoprotein LFA sensor. A further experimentation with clinical samples, we tested it half-stick was prepared by dispensing the R8 antibody on the using artificial saliva, taking into account that COVID-19 test line (TL) and the secondary antibodies (anti-mouse IgG) diagnosis is possible using such a kind of sample.4,38 The on the control line (CL), while the antibody nucleoprotein M5 2940 | Lab Chip, 2022, 22, 2938–2943 This journal is © The Royal Society of Chemistry 2022 View Article Online Lab on a Chip Communication was conjugated to AuNPs. In order to characterise the sensor platform's needs, e.g. antibodies with fast binding kinetics for response, we challenged it using nucleoprotein-spiked a LFA, which features a short receptor–analyte interaction time. artificial saliva samples covering a nucleoprotein We realise that longer bioreceptor characterisation implies concentration range between 1 ng mL−1 (21.2 pM) and 10 μg higher costs for the company (e.g. new instrumentation, This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. mL−1 (0.2 μM) (Fig. 2C). Through the analysis of the obtained delayed commercial availability). Nonetheless, we truly believe half-sticks, we calculated a limit of detection (LoD) of 3.0 ± that researchers would rather buy more expensive, but well 1.2 ng mL−1, limit of quantification (LoQ) of 14.7 ± 2.0 ng characterised antibodies than cheaper but poorly characterised mL−1, and a useful dynamic range of 11.4–791.0 ng mL−1 ones. This in turn would optimise the time and economic (Fig. 2C). The test line signals obtained using nucleoprotein resources required for the development of a point-of-care concentrations higher than 10 ng mL−1 were clearly seen by diagnostic device, speeding up its placement in the market. the naked eye (Fig. 2D). In addition, the recovery of Open Access Article. Published on 29 July 2022. Downloaded on 11/2/2022 7:48:10 AM. nucleoprotein samples ranged from 83.0 ± 2.0% to 116.7 ± Author contributions 16.7%, demonstrating the accuracy of the test (Table S2†). The analytical performance of the described half-sticks is LH: conceptualisation, investigation, formal analysis, comparable to that of a full SARS-CoV-2 nucleoprotein LFA software, and writing – original draft. EC: conceptualisation, recently published for the detection of the nucleoprotein.39 investigation, and writing – original draft. CFC: investigation To further support our results, we found another recently and writing – review and editing. CP: methodology, data published study by Cate et al., in which 1021 anti- curation, supervision, and writing – review and editing. AI: nucleoprotein antibodies were tested in LFA, taking supervision and writing – review and editing. RA: software, advantage of an automatized, high-throughput robotic data curation, and supervision. LR: supervision and writing – system,27 and the couple of antibodies we independently review and editing. AM: project administration, funding selected has been found among the top performing couples. acquisition, and supervision. Finally, to make stronger our message that knowing beforehand the binding behaviour of bioreceptors would Conflicts of interest speed up the development of diagnostic tests, we studied the There are no conflicts to declare. binding constant of three couples of antibodies selected according to their performance in the preliminary steps, Acknowledgements which goes as follows. One of them worked in ELISA but did not in half-stick (R8/M2), while the other two couples did We acknowledge Consejo Superior de Investigaciones work in both types of assays (R8/M4 and R8/M5). R8/M2 Científicas (CSIC) for the project “COVID19-122” granted in showed an EC50 at least 72% higher than those of the other the call “Nuevas ayudas extraordinarias a proyectos de two couples (R8/M4 and R8/M5) and also a higher working investigación en el marco de las medidas urgentes range, thus indicating a lower affinity, which is not suitable extraordinarias para hacer frente al impacto económico y for LFAs (Table S3 and Fig. S4†). social del COVID-19 (Ayudas CSIC-COVID-19)”. We acknowledge also the MICROB-PREDICT project that has Conclusions received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement Despite the successful implementation of the LFA development No. 825694. Financial support from the EU Graphene Flagship protocol,18 the significant amount of invested economic Core 3 Project (No. 881603) is also acknowledged. This article resources (∼25 000 €), personnel, and time (over 10 months) to reflects only the authors' view, and the European Commission identify working antibodies is alarming. We understand that is not responsible for any use that may be made of the the majority of the antibodies are validated for a few specific information it contains. ICN2 is funded by the CERCA applications, generally traditional laboratory-bound techniques programme/Generalitat de Catalunya. ICN2 is supported by (e.g. ELISA, western blot). However, the COVID-19 pandemic the Severo Ochoa Centres of Excellence programme, funded has demonstrated that we cannot rely exclusively on long by the Spanish Research Agency (AEI, grant no. SEV-2017- (hours) and cumbersome (multistep) diagnostic techniques to 0706). E. C. acknowledges Ministerio de Ciencia e Innovación effectively diagnose infectious diseases because we need of Spain and Fondo Social Europeo for the Fellowship sensing platforms able to support massive (or high-frequency) PRE2018-084856 awarded under the call ‘Ayudas para testing. Therefore, we urge antibody producers and distributors contratos predoctorales para la formación de doctores, to consider the implementation of more extensive Subprograma Estatal de Formación del Programa Estatal de characterisation of their products, which would allow Promoción del Talento y su Empleabilidad en I+D+i’, under researchers to make better-informed purchases. At the same the framework of ‘Plan Estatal de Investigación Científica y time, the integration of new antibodies into point-of-care Técnica y de Innovación 2017–2020’. L. H. acknowledges the devices would also be faster. The availability of information China Scholarship Council. L. H., E. C. and C. F.-C. such as the binding kinetics of bioreceptors could lead acknowledge the Autonomous University of Barcelona (UAB) researchers towards purchasing antibodies that better suit their for their support. C. P. (ISGlobal) also acknowledges support This journal is © The Royal Society of Chemistry 2022 Lab Chip, 2022, 22, 2938–2943 | 2941 View Article Online Communication Lab on a Chip from the Spanish Ministry of Science and Innovation and State 17 A. Sena-Torralba, H. Torné-Morató, C. Parolo, S. Ranjbar, Research Agency through the “Centro de Excelencia Severo M. A. Farahmand Nejad, R. Álvarez-Diduk, A. Idili, M. R. Ochoa 2019–2023” Program (CEX2018-000806-S), and support Hormozi-Nezhad and A. Merkoçi, Adv. Mater. Technol., from the Generalitat de Catalunya through the CERCA 2022, 2101450. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Program. A. I. was supported by a PROBIST postdoctoral 18 C. Parolo, A. Sena-Torralba, J. F. 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This journal is © The Royal Society of Chemistry 2022 Lab Chip, 2022, 22, 2938–2943 | 2943