Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets.
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We acknowledge the MICROB-PREDICT project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 825694. Financial support from the EU Graphene Flagship Core 2 Project (No. 785219) is also acknowledged. This article reflects only the author’s view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). C.P. acknowledges the Marie Skłodowska-Curie Actions Individual Fellowship; this project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 795635. E.C. acknowledges Ministerio de Ciencia e Innovación of Spain and Fondo Social Europeo for the Fellowship PRE2018-084856 awarded under the call ‘Ayudas para contratos predoctorales para la formación de doctores, Subprograma Estatal de Formación del Programa Estatal de Promoción del Talento y su Empleabilidad en I+D+i’, under the framework of ‘Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020’. E.P.N. acknowledges funding through the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510. A.M. acknowledges all previous members of the group who have been contributing in the research done on LFAs.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Claudio Baggiani, Daniel T. Kamei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol:
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Parolo, C. et al. Lab Chip 13, 386–390 (2013): https://pubs.rsc.org/en/content/articlelanding/2013/LC/C2LC41144J
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López-Marzo, A. M. et al. Biosens. Bioelectron. 47, 190–198 (2013): https://www.sciencedirect.com/science/article/pii/S0956566313001292
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Parolo, C., Sena-Torralba, A., Bergua, J.F. et al. Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays. Nat Protoc 15, 3788–3816 (2020). https://doi.org/10.1038/s41596-020-0357-x
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