Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays

Abstract

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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Fig. 1: Schematic of the main components and operation of a typical LFA.
Fig. 2: Examples of optical readouts of LFAs using different types of nanoparticles.
Fig. 3: Example results of a gold aggregation test for 20-nm diameter AuNPs and anti-human IgG.
Fig. 4: Step-by-step fabrication of an LFA for the detection of human IgG.
Fig. 5: Different types of readouts of LFAs.
Fig. 6: Qualitative analysis of LFAs for the detection of human IgG.
Fig. 7: Quantitative analysis of an LFA using ImageJ and fitting the results to a four-parameter logistic curve (sigmoidal curve).
Fig. 8: Ideal optimization route for the fabrication of an LFA.

Data availability

The datasets generated during and/or analyzed during the current study (Figs. 3 and 7) are available from the corresponding author on reasonable request.

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Acknowledgements

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.

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C.P. and A.S.-T. designed, organized and wrote the whole manuscript, carried out the experiments, analyzed the data and prepared the figures. J.F.B. wrote the sample pad section. E.C. wrote the nanoparticle section. C.F.-C. wrote the type of sample section. L.H. wrote the membrane section and part of the procedure. L.R. wrote the conjugate pad, Fusion 5 and alternative material sections. R.A.-D. wrote the assay evaluation section and prepared the figures. E.P.N. wrote and edited the manuscript. S.C. wrote the future direction and electrochemical readout sections and helped with the conceptualization. D.Q.-C. wrote the cost, patent, production, regulation and approval sections. A.M. supervised the work.

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Correspondence to Arben Merkoçi.

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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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Related links

Key references using this protocol:

Parolo, C. et al. Biosens. Bioelectron. 40, 412–416 (2013): https://www.sciencedirect.com/science/article/pii/S0956566312004083

Parolo, C. et al. Lab Chip 13, 386–390 (2013): https://pubs.rsc.org/en/content/articlelanding/2013/LC/C2LC41144J

Rivas, L. et al. Lab Chip 14, 4406–4414 (2014): https://pubs.rsc.org/en/content/articlelanding/2014/LC/C4LC00972J

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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