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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1.

    Parolo, C. & Merkoçi, A. Paper-based nanobiosensors for diagnostics. Chem. Soc. Rev. 42, 450–457 (2013).

    CAS  PubMed  Google Scholar 

  2. 2.

    Bahadır, E. B. & Sezgintürk, M. K. Lateral flow assays: principles, designs and labels. Trends Analyt. Chem. 82, 286–306 (2016).

    Google Scholar 

  3. 3.

    Brangel, P. et al. A serological point-of-care test for the detection of IgG antibodies against Ebola virus in human survivors. ACS Nano 12, 63–73 (2018).

    CAS  PubMed  Google Scholar 

  4. 4.

    Posthuma-Trumpie, G. A., Korf, J. & van Amerongen, A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 393, 569–582 (2009).

    CAS  PubMed  Google Scholar 

  5. 5.

    Hu, J. et al. Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 54, 585–597 (2014).

    CAS  PubMed  Google Scholar 

  6. 6.

    Yetisen, A. K., Akram, M. S. & Lowe, C. R. Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 (2013).

    CAS  PubMed  Google Scholar 

  7. 7.

    Martinelli, F. et al. Advanced methods of plant disease detection. A review. Agron. Sustain. Dev 35, 1–25 (2015).

    Google Scholar 

  8. 8.

    Li, P., Zhang, Q. & Zhang, W. Immunoassays for aflatoxins. Trends Analyt. Chem. 28, 1115–1126 (2009).

    CAS  Google Scholar 

  9. 9.

    Anfossi, L., Baggiani, C., Giovannoli, C., D’Arco, G. & Giraudi, G. Lateral-flow immunoassays for mycotoxins and phycotoxins: a review. Anal. Bioanal. Chem. 405, 467–480 (2013).

    CAS  PubMed  Google Scholar 

  10. 10.

    Luo, K., Kim, H.-Y., Oh, M.-H. & Kim, Y.-R. Paper-based lateral flow strip assay for the detection of foodborne pathogens: principles, applications, technological challenges and opportunities. Crit. Rev. Food Sci. Nutr. 60, 157–170 (2020).

    CAS  PubMed  Google Scholar 

  11. 11.

    Ahmed, F. E. Detection of genetically modified organisms in foods. Trends Biotechnol. 20, 215–223 (2002).

    CAS  PubMed  Google Scholar 

  12. 12.

    Ngom, B., Guo, Y., Wang, X. & Bi, D. Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review. Anal. Bioanal. Chem. 397, 1113–1135 (2010).

    CAS  Google Scholar 

  13. 13.

    Zhao, X., Lin, C.-W., Wang, J. & Oh, D. H. Advances in rapid detection methods for foodborne pathogens. J. Microbiol. Biotechnol. 24, 297–312 (2014).

    CAS  PubMed  Google Scholar 

  14. 14.

    Ramage, J. G. et al. Comprehensive laboratory evaluation of a specific lateral flow assay for the presumptive identification of abrin in suspicious white powders and environmental samples. Biosecur. Bioterror. 12, 49–62 (2014).

    PubMed  Google Scholar 

  15. 15.

    Grubb, A. O. & Glad, U. C. Immunoassay with test strip having antibodies bound thereto. US Patent 4,168,146 filed 15 January 1976 and issued 18 September 1979.

  16. 16.

    Hsieh, H. V., Dantzler, J. L. & Weigl, B. H. Analytical tools to improve optimization procedures for lateral flow assays. Diagnostics 7, 29 (2017).

    CAS  PubMed Central  Google Scholar 

  17. 17.

    Gasperino, D., Baughman, T., Hsieh, H. V., Bell, D. & Weigl, B. H. Improving lateral flow assay performance using computational modeling. Annu. Rev. Anal. Chem. 11, 219–244 (2018).

    Google Scholar 

  18. 18.

    Merck Millipore. Rapid lateral flow test strips: considerations for product development. http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-RU-Site/ru_RU/-/USD/ShowDocument-Pronet?id=201306.15671 (2013; accessed 28 July 2020).

  19. 19.

    nanoComposix. Lateral flow assay development guide. https://nanocomposix.com/pages/lateral-flow-assay-development-guide (2018; accessed 28 July 2020).

  20. 20.

    Wong, R. C. & Tse, H. Y., eds. Lateral Flow Immunoassay (Humana Press, 2009).

  21. 21.

    Bishop, J. D., Hsieh, H. V., Gasperino, D. J. & Weigl, B. H. Sensitivity enhancement in lateral flow assays: a systems perspective. Lab Chip 19, 2486–2499 (2019).

    CAS  PubMed  Google Scholar 

  22. 22.

    Zhang, G., Guo, J. & Wang, X. Immunochromatographic lateral flow strip tests. In Biosensors and Biodetection. Methods in Molecular Biology (eds. Rasooly, A. & Herold, K. E.) 169–183 (Humana Press, 2009).

  23. 23.

    Volkov, A., Mauk, M., Corstjens, P. & Niedbala, R. S. Rapid prototyping of lateral flow assays. In Biosensors and Biodetection. Methods in Molecular Biology (eds. Rasooly, A. & Herold, K. E.) 217–235 (Humana Press, 2009).

  24. 24.

    Bailes, J., Mayoss, S., Teale, P. & Soloviev, M. Gold nanoparticle antibody conjugates for use in competitive lateral flow assays. In Nanoparticles in Biology and Medicine. Methods in Molecular Biology (Methods and Protocols) Vol. 906 (ed. Soloviev, M.) 45–55 (Humana Press, 2012).

  25. 25.

    Zeng, L., Lie, P., Fang, Z. & Xiao, Z. Lateral flow biosensors for the detection of nucleic acid. In Nucleic Acid Detection. Methods in Molecular Biology (Methods and Protocols) Vol. 1039 (eds. Kolpashchikov, D. & Gerasimova, Y.) 161–167 (Humana Press, 2013).

  26. 26.

    Ching, K. H. Lateral flow immunoassay. in ELISA: Methods in Molecular Biology (ed. Hnasko, R.) 127–137 (Humana Press, 2015).

  27. 27.

    Tang, R. H. et al. Advances in paper-based sample pretreatment for point-of-care testing. Crit. Rev. Biotechnol. 37, 411–428 (2017).

    CAS  PubMed  Google Scholar 

  28. 28.

    Wang, Y. et al. A SERS-based lateral flow assay biosensor for quantitative and ultrasensitive detection of interleukin-6 in unprocessed whole blood. Biosens. Bioelectron. 141, 111432 (2019).

    CAS  PubMed  Google Scholar 

  29. 29.

    Ang, S. H., Rambeli, M., Thevarajah, T. M., Alias, Y. B. & Khor, S. M. Quantitative, single-step dual measurement of hemoglobin A1c and total hemoglobin in human whole blood using a gold sandwich immunochromatographic assay for personalized medicine. Biosens. Bioelectron. 78, 187–193 (2016).

    CAS  PubMed  Google Scholar 

  30. 30.

    Gao, X. et al. Paper-based surface-enhanced raman scattering lateral flow strip for detection of neuron-specific enolase in blood plasma. Anal. Chem. 89, 10104–10110 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Dieplinger, B., Egger, M., Gegenhuber, A., Haltmayer, M. & Mueller, T. Analytical and clinical evaluation of a rapid quantitative lateral flow immunoassay for measurement of soluble ST2 in human plasma. Clin. Chim. Acta 451, 310–315 (2015).

    CAS  PubMed  Google Scholar 

  32. 32.

    Ou, L. et al. Development of a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific IgM in human serum specimens. J. Microbiol. Methods 124, 35–40 (2016).

    CAS  PubMed  Google Scholar 

  33. 33.

    Huang, Y. et al. Development of up-converting phosphor technology-based lateral flow assay for quantitative detection of serum PIVKA-II: inception of a near-patient PIVKA-II detection tool. Clin. Chim. Acta 488, 202–208 (2019).

    CAS  PubMed  Google Scholar 

  34. 34.

    Chamorro-Garcia, A. et al. Detection of parathyroid hormone-like hormone in cancer cell cultures by gold nanoparticle-based lateral flow immunoassays. Nanomedicine 12, 53–61 (2016).

    CAS  PubMed  Google Scholar 

  35. 35.

    Dalirirad, S. & Steckl, A. J. Aptamer-based lateral flow assay for point of care cortisol detection in sweat. Sens. Actuators B Chem. 283, 79–86 (2019).

    CAS  Google Scholar 

  36. 36.

    Hudson, M. et al. Drug screening using the sweat of a fingerprint: lateral flow detection of Δ9-tetrahydrocannabinol, cocaine, opiates and amphetamine. J. Anal. Toxicol. 43, 88–95 (2019).

    CAS  PubMed  Google Scholar 

  37. 37.

    Dalirirad, S. & Steckl, A. J. Lateral flow assay using aptamer-based sensing for on-site detection of dopamine in urine. Anal. Biochem. 596, 113637 (2020).

    CAS  PubMed  Google Scholar 

  38. 38.

    Li, Z., Chen, H., Feng, S., Liu, K. & Wang, P. Development and clinical validation of a sensitive lateral flow assay for rapid urine fentanyl screening in the emergency department. Clin. Chem. 66, 324–332 (2020).

    PubMed  Google Scholar 

  39. 39.

    Henderson, W. A. et al. Simple lateral flow assays for microbial detection in stool. Anal. Methods 10, 5358–5363 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Lin, Z. et al. Development of an immunochromatographic lateral flow device for rapid detection of Helicobacter pylori stool antigen. Clin. Biochem. 48, 1298–1303 (2015).

    CAS  PubMed  Google Scholar 

  41. 41.

    Hu, Q. et al. An up-converting phosphor technology-based lateral flow assay for point-of-collection detection of morphine and methamphetamine in saliva. Analyst 143, 4646–4654 (2018).

    CAS  PubMed  Google Scholar 

  42. 42.

    Oh, H.-K., Kim, J.-W., Kim, J.-M. & Kim, M.-G. High sensitive and broad-range detection of cortisol in human saliva using a trap lateral flow immunoassay (trapLFI) sensor. Analyst 143, 3883–3889 (2018).

    CAS  PubMed  Google Scholar 

  43. 43.

    Boulware, D. R. et al. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast. Emerg. Infect. Dis. 20, 45–53 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Fleury, A. et al. A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid. Exp. Parasitol. 171, 67–70 (2016).

    CAS  Google Scholar 

  45. 45.

    Sakurai, A. et al. Multi-colored immunochromatography using nanobeads for rapid and sensitive typing of seasonal influenza viruses. J. Virol. Methods 209, 62–68 (2014).

    CAS  PubMed  Google Scholar 

  46. 46.

    Eryılmaz, M. et al. SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique. Analyst 144, 3573–3580 (2019).

    PubMed  Google Scholar 

  47. 47.

    Principato, M. et al. Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays. J. Food Sci. 75, T141–T147 (2010).

    CAS  PubMed  Google Scholar 

  48. 48.

    Jiang, H. et al. Silver nanoparticle-based fluorescence-quenching lateral flow immunoassay for sensitive detection of ochratoxin A in grape juice and wine. Toxins (Basel) 9, 83 (2017).

    Google Scholar 

  49. 49.

    Kong, D. et al. Ultrasensitive and eco-friendly immunoassays based monoclonal antibody for detection of deoxynivalenol in cereal and feed samples. Food Chem. 270, 130–137 (2019).

    CAS  PubMed  Google Scholar 

  50. 50.

    Focker, M., van der Fels-Klerx, H. J. & Oude Lansink, A. G. J. M. Cost-effective sampling and analysis for mycotoxins in a cereal batch. Risk Anal. 39, 926–939 (2019).

    CAS  PubMed  Google Scholar 

  51. 51.

    Yang, X. et al. A lateral flow immunochromato-graphic strip test for rapid detection of oseltamivir phosphate in egg and chicken meat. Sci. Rep. 8, 16680 (2018).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Magiati, M., Myridaki, V. M., Christopoulos, T. K. & Kalogianni, D. P. Lateral flow test for meat authentication with visual detection. Food Chem. 274, 803–807 (2019).

    CAS  PubMed  Google Scholar 

  53. 53.

    Tan, G. et al. Ultrasensitive quantitation of imidacloprid in vegetables by colloidal gold and time-resolved fluorescent nanobead traced lateral flow immunoassays. Food Chem. 311, 126055 (2020).

    CAS  PubMed  Google Scholar 

  54. 54.

    Hassan, A. H. A., Bergua, J. F., Morales-Narváez, E. & Mekoçi, A. Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water. Food Chem. 297, 124965 (2019).

    CAS  PubMed  Google Scholar 

  55. 55.

    Liu, Y. et al. Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay. Anal. Biochem. 483, 7–11 (2015).

    CAS  PubMed  Google Scholar 

  56. 56.

    Wu, Z. et al. Aptamer-based fluorescence-quenching lateral flow strip for rapid detection of mercury (II) ion in water samples. Anal. Bioanal. Chem. 409, 5209–5216 (2017).

    CAS  PubMed  Google Scholar 

  57. 57.

    Quesada-González, D., Jairo, G. A., Blake, R. C., Blake, D. A. & Merkoçi, A. Uranium (VI) detection in groundwater using a gold nanoparticle/paper-based lateral flow device. Sci. Rep 8, 16157 (2018).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Mosley, G. L. et al. Improved lateral-flow immunoassays for chlamydia and immunoglobulin M by sequential rehydration of two-phase system components within a paper-based diagnostic. Microchim. Acta 184, 4055–4064 (2017).

    CAS  Google Scholar 

  59. 59.

    Ridgway, K., Lalljie, S. P. D. & Smith, R. M. Sample preparation techniques for the determination of trace residues and contaminants in foods. J. Chromatogr. A 1153, 36–53 (2007).

    CAS  PubMed  Google Scholar 

  60. 60.

    Gong, M. M., Macdonald, B. D., Vu Nguyen, T., Van Nguyen, K. & Sinton, D. Field tested milliliter-scale blood filtration device for point-of-care applications. Biomicrofluidics 7, 44111 (2013).

    PubMed  Google Scholar 

  61. 61.

    Golden, A. et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS ONE 8, e69231 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Sastre, P. et al. Development of a novel lateral flow assay for detection of African swine fever in blood. BMC Vet. Res. 12, 206 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Choi, J. R. et al. Sensitive biomolecule detection in lateral flow assay with a portable temperature–humidity control device. Biosens. Bioelectron. 79, 98–107 (2016).

    CAS  PubMed  Google Scholar 

  64. 64.

    Fukushi, S. et al. Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA. J. Virol. Methods 251, 22–29 (2018).

    CAS  PubMed  Google Scholar 

  65. 65.

    Duo, J. et al. Surface plasmon resonance as a tool for ligand-binding assay reagent characterization in bioanalysis of biotherapeutics. Bioanalysis 10, 559–576 (2018).

    CAS  PubMed  Google Scholar 

  66. 66.

    Miller, B. S. et al. Quantifying biomolecular binding constants using video paper analytical devices. Chemistry 24, 9783–9787 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Dam, T. K., Torres, M., Brewer, C. F. & Casadevall, A. Isothermal titration calorimetry reveals differential binding thermodynamics of variable region-identical antibodies differing in constant region for a univalent ligand. J. Biol. Chem. 283, 31366–31370 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Yang, D., Singh, A., Wu, H. & Kroe-Barrett, R. Determination of high-affinity antibody-antigen binding kinetics using four biosensor platforms. J. Vis. Exp. e55659 (2017).

  69. 69.

    Mosley, G. L., Nguyen, P., Wu, B. M. & Kamei, D. T. Development of quantitative radioactive methodologies on paper to determine important lateral-flow immunoassay parameters. Lab Chip 16, 2871–2881 (2016).

    CAS  PubMed  Google Scholar 

  70. 70.

    Wang, C. et al. Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk. J. Dairy Sci. 101, 8767–8777 (2018).

    CAS  PubMed  Google Scholar 

  71. 71.

    Nguyen, V.-T., Song, S., Park, S. & Joo, C. Recent advances in high-sensitivity detection methods for paper-based lateral-flow assay. Biosens. Bioelectron. 152, 112015 (2020).

    CAS  PubMed  Google Scholar 

  72. 72.

    Quesada-González, D. & Merkoçi, A. Nanoparticle-based lateral flow biosensors. Biosens. Bioelectron. 73, 47–63 (2015).

    PubMed  Google Scholar 

  73. 73.

    Soh, J. H., Chan, H.-M. & Ying, J. Y. Strategies for developing sensitive and specific nanoparticle-based lateral flow assays as point-of-care diagnostic device. Nano Today 30, 100831 (2020).

    CAS  Google Scholar 

  74. 74.

    Ge, X. et al. Nanomaterial-enhanced paper-based biosensors. Trends Analyt. Chem. 58, 31–39 (2014).

    CAS  Google Scholar 

  75. 75.

    Zhan, L. et al. The role of nanoparticle design in determining analytical performance of lateral flow immunoassays. Nano Lett. 17, 7207–7212 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Quesada-González, D. & Merkoçi, A. Nanomaterial-based devices for point-of-care diagnostic applications. Chem. Soc. Rev. 47, 4697–4709 (2018).

    PubMed  Google Scholar 

  77. 77.

    Verheijen, R., Osswald, I. K., Dietrich, R. & Haasnoot, W. Development of a one step strip test for the detection of (dihydro)streptomycin residues in raw milk. Food Agric. Immunol. 12, 31–40 (2000).

    CAS  Google Scholar 

  78. 78.

    Fong, W. K. et al. Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology. J. Clin. Microbiol 38, 2525–2529 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Shyu, R. H., Shyu, H. F., Liu, H. W. & Tang, S. S. Colloidal gold-based immunochromatographic assay for detection of ricin. Toxicon 40, 255–258 (2002).

    CAS  PubMed  Google Scholar 

  80. 80.

    Ren, W., Ballou, D. R., FitzGerald, R. & Irudayaraj, J. Plasmonic enhancement in lateral flow sensors for improved sensing of E. coli O157:H7. Biosens. Bioelectron. 126, 324–331 (2019).

    CAS  PubMed  Google Scholar 

  81. 81.

    Innova Biosciences. Guide to lateral flow immunoassays: Innova Biosciences guide. https://fnkprddata.blob.core.windows.net/domestic/download/pdf/IBS_A_guide_to_lateral_flow_immunoassays.pdf (2020; accessed 28 July 2020).

  82. 82.

    Di Nardo, F., Cavalera, S., Baggiani, C., Giovannoli, C. & Anfossi, L. Direct vs mediated coupling of antibodies to gold nanoparticles: the case of salivary cortisol detection by lateral flow immunoassay. ACS Appl. Mater. Interfaces 11, 32758–32768 (2019).

    PubMed  Google Scholar 

  83. 83.

    Parolo, C. et al. Design, preparation, and evaluation of a fixed-orientation antibody/gold-nanoparticle conjugate as an immunosensing label. ACS Appl. Mater. Interfaces 5, 10753–10759 (2013).

    CAS  PubMed  Google Scholar 

  84. 84.

    Liu, J., Mazumdar, D. & Lu, Y. A simple and sensitive ‘dipstick’ test in serum based on lateral flow separation of aptamer-linked nanostructures. Angew. Chem. Int. Ed. Engl. 45, 7955–7959 (2006).

    CAS  PubMed  Google Scholar 

  85. 85.

    Mao, X. et al. Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip. Anal. Chem. 81, 1660–1668 (2009).

    CAS  PubMed  Google Scholar 

  86. 86.

    Posthuma-Trumpie, G. A., Wichers, J. H., Koets, M., Berendsen, L. B. J. M. & van Amerongen, A. Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays. Anal. Bioanal. Chem. 402, 593–600 (2012).

    CAS  PubMed  Google Scholar 

  87. 87.

    Van Dam, G. J. et al. Diagnosis of schistosomiasis by reagent strip test for detection of circulating cathodic antigen. J. Clin. Microbiol. 42, 5458–5461 (2004).

    PubMed  PubMed Central  Google Scholar 

  88. 88.

    Blazková, M., Micková-Holubová, B., Rauch, P. & Fukal, L. Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water. Biosens. Bioelectron. 25, 753–758 (2009).

    PubMed  Google Scholar 

  89. 89.

    Blažková, M., Rauch, P. & Fukal, L. Strip-based immunoassay for rapid detection of thiabendazole. Biosens. Bioelectron. 25, 2122–2128 (2010).

    PubMed  Google Scholar 

  90. 90.

    Kalogianni, D. P., Boutsika, L. M., Kouremenou, P. G., Christopoulos, T. K. & Ioannou, P. C. Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization. Anal. Bioanal. Chem. 400, 1145–1152 (2011).

    CAS  PubMed  Google Scholar 

  91. 91.

    Noguera, P. et al. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Anal. Bioanal. Chem. 399, 831–838 (2011).

    CAS  PubMed  Google Scholar 

  92. 92.

    Oliveira-Rodríguez, M. et al. Point-of-care detection of extracellular vesicles: sensitivity optimization and multiple-target detection. Biosens. Bioelectron. 87, 38–45 (2017).

    PubMed  Google Scholar 

  93. 93.

    Qiu, W. et al. Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence. Biosens. Bioelectron. 64, 367–372 (2015).

    CAS  PubMed  Google Scholar 

  94. 94.

    Yao, L. et al. MWCNTs based high sensitive lateral flow strip biosensor for rapid determination of aqueous mercury ions. Biosens. Bioelectron. 85, 331–336 (2016).

    CAS  PubMed  Google Scholar 

  95. 95.

    Greenwald, R. et al. Improved serodetection of Mycobacterium bovis infection in badgers (Meles meles) using multiantigen test formats. Diagn. Microbiol. Infect. Dis. 46, 197–203 (2003).

    CAS  PubMed  Google Scholar 

  96. 96.

    Morales-Narváez, E., Naghdi, T., Zor, E. & Merkoçi, A. Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection. Anal. Chem. 87, 8573–8577 (2015).

    PubMed  Google Scholar 

  97. 97.

    Medintz, I. L., Uyeda, T. H., Goldman, E. R. & Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446 (2005).

    CAS  PubMed  Google Scholar 

  98. 98.

    Taranova, N. A., Berlina, A. N., Zherdev, A. V. & Dzantiev, B. B. ‘Traffic light’ immunochromatographic test based on multicolor quantum dots for the simultaneous detection of several antibiotics in milk. Biosens. Bioelectron. 63, 255–261 (2015).

    CAS  PubMed  Google Scholar 

  99. 99.

    Foubert, A. et al. Development of a rainbow lateral flow immunoassay for the simultaneous detection of four mycotoxins. J. Agric. Food Chem. 65, 7121–7130 (2017).

    CAS  PubMed  Google Scholar 

  100. 100.

    Wang, C. et al. Layer-by-layer assembly of magnetic-core dual quantum dot-shell nanocomposites for fluorescence lateral flow detection of bacteria. Nanoscale 12, 795–807 (2020).

    CAS  PubMed  Google Scholar 

  101. 101.

    Yan, X. et al. CdSe/ZnS quantum dot-labeled lateral flow strips for rapid and quantitative detection of gastric cancer carbohydrate antigen 72-4. Nanoscale Res. Lett. 11, 138 (2016).

    PubMed  PubMed Central  Google Scholar 

  102. 102.

    Berlina, A. N., Taranova, N. A., Zherdev, A. V., Vengerov, Y. Y. & Dzantiev, B. B. Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk. Anal. Bioanal. Chem. 405, 4997–5000 (2013).

    CAS  PubMed  Google Scholar 

  103. 103.

    Bruno, J. G. Application of DNA aptamers and quantum dots to lateral flow test strips for detection of foodborne pathogens with improved sensitivity versus colloidal gold. Pathogens 3, 341–355 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104.

    Bruno, J. G. Evaluation of pathogenic big 7 E. coli aptamer-quantum dot lateral flow test strips. J. Bionanoscience 11, 148–152 (2017).

    CAS  Google Scholar 

  105. 105.

    Yang, H. et al. A novel quantum dots-based point of care test for syphilis. Nanoscale Res. Lett. 5, 875–881 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Zamora-Gálvez, A., Morales-Narváez, E., Romero, J. & Merkoçi, A. Photoluminescent lateral flow based on non-radiative energy transfer for protein detection in human serum. Biosens. Bioelectron. 100, 208–213 (2018).

    PubMed  Google Scholar 

  107. 107.

    Chen, J. et al. A facile fluorescence lateral flow biosensor for glutathione detection based on quantum dots-MnO2 nanocomposites. Sens. Actuators B Chem. 260, 770–777 (2018).

    CAS  Google Scholar 

  108. 108.

    Rong, Z. et al. Dual-color magnetic-quantum dot nanobeads as versatile fluorescent probes in test strip for simultaneous point-of-care detection of free and complexed prostate-specific antigen. Biosens. Bioelectron. 145, 111719 (2019).

    CAS  PubMed  Google Scholar 

  109. 109.

    Niedbala, R. S. et al. Detection of analytes by immunoassay using up-converting phosphor technology. Anal. Biochem. 293, 22–30 (2001).

    CAS  PubMed  Google Scholar 

  110. 110.

    Corstjens, P. et al. Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection. Clin. Chem. 47, 1885–1893 (2001).

    CAS  PubMed  Google Scholar 

  111. 111.

    Hampl, J. et al. Upconverting phosphor reporters in immunochromatographic assays. Anal. Biochem. 288, 176–187 (2001).

    CAS  PubMed  Google Scholar 

  112. 112.

    Kim, J. et al. Rapid and background-free detection of avian influenza virus in opaque sample using NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay platform. Biosens. Bioelectron. 112, 209–215 (2018).

    CAS  Google Scholar 

  113. 113.

    He, H. et al. Quantitative lateral flow strip sensor using highly doped upconversion nanoparticles. Anal. Chem. 90, 12356–12360 (2018).

    CAS  PubMed  Google Scholar 

  114. 114.

    You, M. et al. Household fluorescent lateral flow strip platform for sensitive and quantitative prognosis of heart failure using dual-color upconversion nanoparticles. ACS Nano 11, 6261–6270 (2017).

    CAS  PubMed  Google Scholar 

  115. 115.

    Gong, Y. et al. A portable and universal upconversion nanoparticle-based lateral flow assay platform for point-of-care testing. Talanta 201, 126–133 (2019).

    CAS  PubMed  Google Scholar 

  116. 116.

    Khreich, N. et al. Detection of Staphylococcus enterotoxin B using fluorescent immunoliposomes as label for immunochromatographic testing. Anal. Biochem. 377, 182–188 (2008).

    CAS  PubMed  Google Scholar 

  117. 117.

    Baeumner, A. J., Jones, C. & Yee, C. A generic sandwich-type biosensor with nanomolar detection limits. Anal. Bioanal. Chem. 378, 1587–1593 (2004).

    CAS  PubMed  Google Scholar 

  118. 118.

    Edwards, K. A. & Baeumner, A. J. Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization. Anal. Bioanal. Chem. 386, 1335–1343 (2006).

    CAS  PubMed  Google Scholar 

  119. 119.

    Edwards, K. A., Korff, R. & Baeumner, A. J. Liposome-enhanced lateral-flow assays for clinical analyses. Methods Mol. Biol. 1571, 407–434 (2017).

    CAS  PubMed  Google Scholar 

  120. 120.

    Urusov, A. E., Zherdev, A. V. & Dzantiev, B. B. Towards lateral flow quantitative assays: detection approaches. Biosensors 9, 89 (2019).

    CAS  PubMed Central  Google Scholar 

  121. 121.

    Yang, J. et al. Detection platforms for point-of-care testing based on colorimetric, luminescent and magnetic assays: a review. Talanta 202, 96–110 (2019).

    CAS  PubMed  Google Scholar 

  122. 122.

    Nash, M. A., Waitumbi, J. N., Hoffman, A. S., Yager, P. & Stayton, P. S. Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system. ACS Nano 6, 6776–6785 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. 123.

    Marquina, C. et al. GMR sensors and magnetic nanoparticles for immuno-chromatographic assays. J. Magn. Magn. Mater. 324, 3495–3498 (2012).

    CAS  Google Scholar 

  124. 124.

    Ryu, Y., Jin, Z., Kang, M. S. & Kim, H. S. Increase in the detection sensitivity of a lateral flow assay for a cardiac marker by oriented immobilization of antibody. Biochip J. 5, 193–198 (2011).

    CAS  Google Scholar 

  125. 125.

    Wang, D. B. et al. Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on ‘Road Closure’. Biosens. Bioelectron. 67, 608–614 (2015).

    CAS  PubMed  Google Scholar 

  126. 126.

    Liu, D. et al. A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations. Food Control 51, 218–224 (2015).

    Google Scholar 

  127. 127.

    Zheng, C., Wang, X., Lu, Y. & Liu, Y. Rapid detection of fish major allergen parvalbumin using superparamagnetic nanoparticle-based lateral flow immunoassay. Food Control 26, 446–452 (2012).

    CAS  Google Scholar 

  128. 128.

    Panferov, V. G., Safenkova, I. V., Zherdev, A. V. & Dzantiev, B. B. Setting up the cut-off level of a sensitive barcode lateral flow assay with magnetic nanoparticles. Talanta 164, 69–76 (2017).

    CAS  PubMed  Google Scholar 

  129. 129.

    Lago-Cachon, D. et al. Scanning magneto-inductive sensor for quantitative assay of prostate-specific antigen. IEEE Magn. Lett 8, 1–5 (2017).

    Google Scholar 

  130. 130.

    Moyano, A. et al. Magnetic immunochromatographic test for histamine detection in wine. Anal. Bioanal. Chem. 411, 6615–6624 (2019).

    CAS  PubMed  Google Scholar 

  131. 131.

    Qin, Z. et al. Significantly improved analytical sensitivity of lateral flow immunoassays by using thermal contrast. Angew. Chem. Int. Ed. Engl. 51, 4358–4361 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132.

    Wang, Y. et al. Thermal contrast amplification reader yielding 8-fold analytical improvement for disease detection with lateral flow assays. Anal. Chem. 88, 11774–11782 (2016).

    CAS  PubMed  Google Scholar 

  133. 133.

    Sinawang, P. D., Rai, V., Ionescu, R. E. & Marks, R. S. Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein. Biosens. Bioelectron. 77, 400–408 (2016).

    CAS  PubMed  Google Scholar 

  134. 134.

    Aller Pellitero, M., Kitsara, M., Eibensteiner, F. & del Campo, F. J. Rapid prototyping of electrochemical lateral flow devices: stencilled electrodes. Analyst 141, 2515–2522 (2016).

    CAS  PubMed  Google Scholar 

  135. 135.

    Ruiz-Vega, G., Kitsara, M., Pellitero, M. A., Baldrich, E. & del Campo, F. J. Electrochemical lateral flow devices: towards rapid immunomagnetic assays. ChemElectroChem 4, 880–889 (2017).

    CAS  Google Scholar 

  136. 136.

    Zhu, X., Shah, P., Stoff, S., Liu, H. & Li, C. A paper electrode integrated lateral flow immunosensor for quantitative analysis of oxidative stress induced DNA damage. Analyst 139, 2850–2857 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. 137.

    Cinti, S., Moscone, D. & Arduini, F. Preparation of paper-based devices for reagentless electrochemical (bio)sensor strips. Nat. Protoc. 14, 2437–2451 (2019).

    CAS  PubMed  Google Scholar 

  138. 138.

    Gonzalez-Macia, L., Morrin, A., Smyth, M. R. & Killard, A. J. Advanced printing and deposition methodologies for the fabrication of biosensors and biodevices. Analyst 135, 845 (2010).

    CAS  PubMed  Google Scholar 

  139. 139.

    Li, Z. et al. Pen-on-paper strategy for point-of-care testing: rapid prototyping of fully written microfluidic biosensor. Biosens. Bioelectron. 98, 478–485 (2017).

    PubMed  Google Scholar 

  140. 140.

    Millipore Sigma. IVD lateral flow: sample, conjugate and absorbent pad basics. https://www.sigmaaldrich.com/technical-documents/articles/ivd-immunoassay/lateral-flow/pads-chemistries-selections-specifications-and-conjugates.html (2020; accessed 28 July 2020).

  141. 141.

    Quesada-González, D. et al. Iridium oxide (IV) nanoparticle-based electrocatalytic detection of PBDE. Biosens. Bioelectron. 127, 150–154 (2019).

    PubMed  Google Scholar 

  142. 142.

    Parolo, C., Medina-Sánchez, M., de la Escosura-Muñiz, A. & Merkoçi, A. Simple paper architecture modifications lead to enhanced sensitivity in nanoparticle based lateral flow immunoassays. Lab Chip 13, 386–390 (2013).

    CAS  PubMed  Google Scholar 

  143. 143.

    Tsai, T.-T. et al. Development a stacking pad design for enhancing the sensitivity of lateral flow immunoassay. Sci. Rep 8, 17319 (2018).

    PubMed  PubMed Central  Google Scholar 

  144. 144.

    Shao, X.-Y. et al. Rapid and sensitive lateral flow immunoassay method for procalcitonin (PCT) based on time-resolved immunochromatography. Sensors 17, 480 (2017).

    Google Scholar 

  145. 145.

    Li, J., McMillan, D. & Macdonal, J. Enhancing the signal of lateral flow immunoassays by using different developing methods. Sensors Mater. 27, 549–561 (2015).

    CAS  Google Scholar 

  146. 146.

    West, M., Walters, F., Phillips, S. & Rowles, D. Enhanced performance of a lateral flow assay: use of a novel conjugate blocking technology to improve performance of a gold nanoparticle-based lateral flow assy (BBI Morffi Whitepaper). https://www.bbisolutions.com/pub/media/wysiwyg/technical_support/BBI_WHITEPAPER_A4_MORFFI_DIGITAL-linked.pdf (2020; accessed 28 July 2020).

  147. 147.

    Phillips, S. Reagent chemistries and labels of choice for lateral flow. https://www.emdmillipore.com/INTERSHOP/static/WFS/Merck-Site/-/Merck/en_US/Freestyle/DIV-Divisional/Events/pdfs/lateral-flow-presentations/reagent-chemistries-and-labels-of-choice-for-lateral-flow.pdf (2020; accessed 28 July 2020).

  148. 148.

    Shim, W.-B., Kim, J.-S., Kim, M.-G. & Chung, D.-H. Rapid and sensitive immunochromatographic strip for on-site detection of sulfamethazine in meats and eggs. J. Food Sci. 78, M1575–M1581 (2013).

    CAS  PubMed  Google Scholar 

  149. 149.

    Yahaya, M. L., Zakaria, N. D., Noordin, R. & Razak, K. A. The effect of nitrocellulose membrane pore size of lateral flow immunoassay on sensitivity for detection of Shigella sp. in milk sample. Mater. Today Proc. 17, 878–883 (2019).

    CAS  Google Scholar 

  150. 150.

    Sartorius Stedim Biotech. UniSart nitrocellulose membranes: the substrate of choice for protein assays. https://www.sartorius.com/resource/blob/89574/dc103586e857d533c5901961867f5ed9/broch-unisart-nitro-sl-1536-e-1-data.pdf (2018; accessed 28 July 2020).

  151. 151.

    Tovey, E. R. & Baldo, B. A. Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting. J. Biochem. Biophys. Methods 19, 169–183 (1989).

    CAS  PubMed  Google Scholar 

  152. 152.

    Asiaei, S., Bidgoli, M. R., ZadehKafi, A., Saderi, N. & Siavashi, M. Sensitivity and colour intensity enhancement in lateral flow immunoassay tests by adjustment of test line position. Clin. Chim. Acta 487, 210–215 (2018).

    CAS  PubMed  Google Scholar 

  153. 153.

    Ragavendar, M. S. & Anmol, C. M. A mathematical model to predict the optimal test line location and sample volume for lateral flow immunoassays. 2012 Annual International Conference of the IEEE Engineering in Medicine and Biology Society 2408–2411 (IEEE, 2012).

  154. 154.

    Fusion 5. Cytiva. https://www.gelifesciences.com/en/us/shop/whatman-laboratory-filtration/whatman-dx-components/lateral-flow-pads/fusion-5-p-00787 (2020; accessed 28 July 2020).

  155. 155.

    Carrell, C. S. et al. Rotary manifold for automating a paper-based Salmonella immunoassay. RSC Adv. 9, 29078–29086 (2019).

    CAS  Google Scholar 

  156. 156.

    Yang, J. J., Oh, H.-B. & Hwang, S.-H. Paper-based speedy separation of amplified DNA (PASS-DNA): potential for molecular point-of-care testing. Sens. Actuators B Chem. 286, 101–103 (2019).

    CAS  Google Scholar 

  157. 157.

    Tang, R. et al. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip 17, 1270–1279 (2017).

    CAS  PubMed  Google Scholar 

  158. 158.

    Nurul Najian, A. B., Engku Nur Syafirah, E. A. R., Ismail, N., Mohamed, M. & Yean, C. Y. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira. Anal. Chim. Acta 903, 142–148 (2016).

    CAS  PubMed  Google Scholar 

  159. 159.

    Chua, A., Yean, C. Y., Ravichandran, M., Lim, B. & Lalitha, P. A rapid DNA biosensor for the molecular diagnosis of infectious disease. Biosens. Bioelectron. 26, 3825–3831 (2011).

    CAS  PubMed  Google Scholar 

  160. 160.

    Choi, D. H. et al. A dual gold nanoparticle conjugate-based lateral flow assay (LFA) method for the analysis of troponin I. Biosens. Bioelectron. 25, 1999–2002 (2010).

    CAS  PubMed  Google Scholar 

  161. 161.

    Hagström, A. E. V. et al. Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay. PLoS ONE 10, e0126571 (2015).

    PubMed  PubMed Central  Google Scholar 

  162. 162.

    Kim, J. et al. Orientational binding modes of reporters in a viral-nanoparticle lateral flow assay. Analyst 142, 55–64 (2017).

    CAS  Google Scholar 

  163. 163.

    Zhou, G., Mao, X. & Juncker, D. Immunochromatographic assay on thread. Anal. Chem. 84, 7736–7743 (2012).

    CAS  PubMed  Google Scholar 

  164. 164.

    Meng, L.-L., Song, T.-T. & Mao, X. Novel immunochromatographic assay on cotton thread based on carbon nanotubes reporter probe. Talanta 167, 379–384 (2017).

    CAS  PubMed  Google Scholar 

  165. 165.

    Jia, X., Song, T., Liu, Y., Meng, L. & Mao, X. An immunochromatographic assay for carcinoembryonic antigen on cotton thread using a composite of carbon nanotubes and gold nanoparticles as reporters. Anal. Chim. Acta 969, 57–62 (2017).

    CAS  PubMed  Google Scholar 

  166. 166.

    Seth, M., Mdetele, D. & Buza, J. Immunochromatographic thread-based test platform for diagnosis of infectious diseases. Microfluid. Nanofluidics 22, 45 (2018).

    Google Scholar 

  167. 167.

    Lappalainen, T., Teerinen, T., Vento, P., Hakalahti, L. & Erho, T. Cellulose as a novel substrate for lateral flow assay. Nord. Pulp Pap. Res. J. 25, 536–550 (2010).

    CAS  Google Scholar 

  168. 168.

    Du, S., Lin, H., Sui, J., Wang, X. & Cao, L. Nano-gold capillary immunochromatographic assay for parvalbumin. Anal. Bioanal. Chem. 406, 6637–6646 (2014).

    CAS  PubMed  Google Scholar 

  169. 169.

    Qu, X. et al. Development of a nano-gold capillary immunochromatographic assay for rapid and semi-quantitative detection of clenbuterol residues. Food Anal. Methods 9, 2531–2540 (2016).

    Google Scholar 

  170. 170.

    Cao, E., Chen, Y., Cui, Z. & Foster, P. R. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol. Bioeng. 82, 684–690 (2003).

    CAS  PubMed  Google Scholar 

  171. 171.

    O’Farrell, B. Lateral flow technology for field-based applications—basics and advanced developments. Top. Companion Anim. Med. 30, 139–147 (2015).

    Google Scholar 

  172. 172.

    Tian, T. et al. Distance-based microfluidic quantitative detection methods for point-of-care testing. Lab Chip 16, 1139–1151 (2016).

    CAS  PubMed  Google Scholar 

  173. 173.

    Leung, W. et al. InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections. J. Immunol. Methods 336, 30–36 (2008).

    CAS  PubMed  Google Scholar 

  174. 174.

    Mak, W. C., Beni, V. & Turner, A. P. F. Lateral-flow technology: from visual to instrumental. Trends Analyt. Chem. 79, 297–305 (2016).

    CAS  Google Scholar 

  175. 175.

    Shah, K. G., Singh, V., Kauffman, P. C., Abe, K. & Yager, P. Mobile phone ratiometric imaging enables highly sensitive fluorescence lateral flow immunoassays without external optical filters. Anal. Chem. 90, 6967–6974 (2018).

    CAS  PubMed  Google Scholar 

  176. 176.

    Zangheri, M. et al. A simple and compact smartphone accessory for quantitative chemiluminescence-based lateral flow immunoassay for salivary cortisol detection. Biosens. Bioelectron. 64, 63–68 (2015).

    CAS  PubMed  Google Scholar 

  177. 177.

    Roda, A. et al. Smartphone-based biosensors: a critical review and perspectives. Trends Anal. Chem. 79, 317–325 (2016).

    CAS  Google Scholar 

  178. 178.

    Jiang, N. et al. Lateral and vertical flow assays for point-of-care diagnostics. Adv. Healthc. Mater. 8, e1900244 (2019).

    PubMed  Google Scholar 

  179. 179.

    de Puig, H., Bosch, I., Gehrke, L. & Hamad-Schifferli, K. Challenges of the nano-bio interface in lateral flow and dipstick immunoassays. Trends Biotechnol. 35, 1169–1180 (2017).

    PubMed  PubMed Central  Google Scholar 

  180. 180.

    Oh, Y. K., Joung, H.-A., Han, H. S., Suk, H.-J. & Kim, M.-G. A three-line lateral flow assay strip for the measurement of C-reactive protein covering a broad physiological concentration range in human sera. Biosens. Bioelectron. 61, 285–289 (2014).

    CAS  PubMed  Google Scholar 

  181. 181.

    Li, J. & Macdonald, J. Multiplexed lateral flow biosensors: technological advances for radically improving point-of-care diagnoses. Biosens. Bioelectron. 83, 177–192 (2016).

    CAS  PubMed  Google Scholar 

  182. 182.

    Gao, Z. et al. Platinum-decorated gold nanoparticles with dual functionalities for ultrasensitive colorimetric in vitro diagnostics. Nano Lett. 17, 5572–5579 (2017).

    CAS  PubMed  Google Scholar 

  183. 183.

    Kim, H., Chung, D.-R. & Kang, M. A new point-of-care test for the diagnosis of infectious diseases based on multiplex lateral flow immunoassays. Analyst 144, 2460–2466 (2019).

    CAS  PubMed  Google Scholar 

  184. 184.

    Mohd Hanafiah, K. et al. Development of multiplexed infectious disease lateral flow assays: challenges and opportunities. Diagnostics 7, 51 (2017).

    PubMed Central  Google Scholar 

  185. 185.

    Qin, Q. et al. Algorithms for immunochromatographic assay: review and impact on future application. Analyst 144, 5659–5676 (2019).

    CAS  PubMed  Google Scholar 

  186. 186.

    Liu, Z. et al. An improved detection limit and working range of lateral flow assays based on a mathematical model. Analyst 143, 2775–2783 (2018).

    CAS  PubMed  Google Scholar 

  187. 187.

    Gantelius, J., Bass, T., Sjöberg, R., Nilsson, P. & Andersson-Svahn, H. A lateral flow protein microarray for rapid and sensitive antibody assays. Int. J. Mol. Sci. 12, 7748–7759 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  188. 188.

    Choi, J. R. et al. An integrated lateral flow assay for effective DNA amplification and detection at the point of care. Analyst 141, 2930–2939 (2016).

    CAS  PubMed  Google Scholar 

  189. 189.

    Jauset-Rubio, M. et al. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Sci. Rep. 6, 37732 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  190. 190.

    Deng, X. et al. Applying strand displacement amplification to quantum dots-based fluorescent lateral flow assay strips for HIV-DNA detection. Biosens. Bioelectron. 105, 211–217 (2018).

    PubMed  Google Scholar 

  191. 191.

    Ivanov, A. V., Safenkova, I. V., Zherdev, A. V. & Dzantiev, B. B. Nucleic acid lateral flow assay with recombinase polymerase amplification: solutions for highly sensitive detection of RNA virus. Talanta 210, 120616 (2020).

    CAS  PubMed  Google Scholar 

  192. 192.

    Lafleur, L. K. et al. A rapid, instrument-free, sample-to-result nucleic acid amplification test. Lab Chip 16, 3777–3787 (2016).

    CAS  PubMed  Google Scholar 

  193. 193.

    Xu, Y. et al. Nucleic acid biosensor synthesis of an all-in-one universal blocking linker recombinase polymerase amplification with a peptide nucleic acid-based lateral flow device for ultrasensitive detection of food pathogens. Anal. Chem. 90, 708–715 (2018).

    CAS  PubMed  Google Scholar 

  194. 194.

    Cheng, N. et al. Specific and relative detection of urinary microRNA signatures in bladder cancer for point-of-care diagnostics. Chem. Commun. 53, 4222–4225 (2017).

    CAS  Google Scholar 

  195. 195.

    Tang, R. et al. Improved sensitivity of lateral flow assay using paper-based sample concentration technique. Talanta 152, 269–276 (2016).

    CAS  PubMed  Google Scholar 

  196. 196.

    Quesada-González, D. et al. Signal enhancement on gold nanoparticle-based lateral flow tests using cellulose nanofibers. Biosens. Bioelectron. 141, 111407 (2019).

    PubMed  Google Scholar 

  197. 197.

    Bai, Y. et al. A sensitive lateral flow test strip based on silica nanoparticle/CdTe quantum dot composite reporter probes. RSC Adv. 2, 1778 (2012).

    CAS  Google Scholar 

  198. 198.

    Zhao, P. et al. Upconversion fluorescent strip sensor for rapid determination of Vibrio anguillarum. Nanoscale 6, 3804–3809 (2014).

    CAS  PubMed  Google Scholar 

  199. 199.

    Ambrosi, A., Airò, F. & Merkoçi, A. Enhanced gold nanoparticle based ELISA for a breast cancer biomarker. Anal. Chem. 82, 1151–1156 (2010).

    CAS  PubMed  Google Scholar 

  200. 200.

    Ford, J. Plasma separation: why do you need it and how do you achieve it. DCN Diagnostics. https://dcndx.com/plasma-separation-why-you-need-it/ (2019; accessed 28 July 2020).

  201. 201.

    Nawattanapaiboon, K. et al. Hemoculture and direct sputum detection of mecA-mediated methicillin-resistant Staphylococcus aureus by loop-mediated isothermal amplification in combination with a lateral-flow dipstick. J. Clin. Lab. Anal. 30, 760–767 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  202. 202.

    Xu, S. et al. Lateral flow immunoassay based on polydopamine-coated gold nanoparticles for the sensitive detection of zearalenone in maize. ACS Appl. Mater. Interfaces 11, 31283–31290 (2019).

    CAS  PubMed  Google Scholar 

  203. 203.

    Anfossi, L. et al. A lateral flow immunoassay for the rapid detection of ochratoxin A in wine and grape must. J. Agric. Food Chem. 60, 11491–11497 (2012).

    CAS  PubMed  Google Scholar 

  204. 204.

    Chen, A. & Yang, S. Replacing antibodies with aptamers in lateral flow immunoassay. Biosens. Bioelectron. 71, 230–242 (2015).

    CAS  PubMed  Google Scholar 

  205. 205.

    Rey, E. G., O’Dell, D., Mehta, S. & Erickson, D. Mitigating the hook effect in lateral flow sandwich immunoassays using real-time reaction kinetics. Anal. Chem. 89, 5095–5100 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  206. 206.

    Wiederhold, N. P. et al. Interlaboratory and interstudy reproducibility of a novel lateral-flow device and influence of antifungal therapy on detection of invasive pulmonary aspergillosis. J. Clin. Microbiol. 51, 459–465 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  207. 207.

    Christau, S., Moeller, T., Genzer, J., Koehler, R. & von Klitzing, R. Salt-induced aggregation of negatively charged gold nanoparticles confined in a polymer brush matrix. Macromolecules 50, 7333–7343 (2017).

    CAS  Google Scholar 

  208. 208.

    Ruiz-Sanchez, A. J. et al. Tuneable plasmonic gold dendrimer nanochains for sensitive disease detection. J. Mater. Chem. B 5, 7262–7266 (2017).

    CAS  PubMed  Google Scholar 

  209. 209.

    Findlay, J. W. A. & Dillard, R. F. Appropriate calibration curve fitting in ligand binding assays. AAPS J. 9, E260–E267 (2007).

    PubMed  PubMed Central  Google Scholar 

  210. 210.

    Holstein, C. A., Griffin, M., Hong, J. & Sampson, P. D. Statistical method for determining and comparing limits of detection of bioassays. Anal. Chem. 87, 9795–9801 (2015).

    CAS  PubMed  Google Scholar 

  211. 211.

    Faber, N. M. The limit of detection is not the analyte level for deciding between “detected” and “not detected”. Accred. Qual. Assur. 13, 277–278 (2008).

    CAS  Google Scholar 

  212. 212.

    Armbruster, D. A. & Pry, T. Limit of blank, limit of detection and limit of quantitation. Clin. Biochem. Rev. 29(Suppl 1), S49–S52 (2008).

    PubMed  PubMed Central  Google Scholar 

  213. 213.

    Wen, H. W., Borejsza-Wysocki, W., Decory, T. R. & Durst, R. A. Development of a competitive liposome-based lateral flow assay for the rapid detection of the allergenic peanut protein Ara h1. Anal. Bioanal. Chem. 382, 1217–1226 (2005).

    CAS  PubMed  Google Scholar 

  214. 214.

    Apilux, A., Rengpipat, S., Suwanjang, W. & Chailapakul, O. Development of competitive lateral flow immunoassay coupled with silver enhancement for simple and sensitive salivary cortisol detection. EXCLI J. 17, 1198–1209 (2018).

    PubMed  PubMed Central  Google Scholar 

  215. 215.

    Posthuma-Trumpie, G. A., Korf, J. & van Amerongen, A. Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components. Anal. Bioanal. Chem. 392, 1215–1223 (2008).

    CAS  PubMed  Google Scholar 

  216. 216.

    Corstjens, P. L. A. M. et al. A user-friendly, highly sensitive assay to detect the IFN-γ secretion by T cells. Clin. Biochem. 41, 440–444 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  217. 217.

    Rivas, L., Medina-Sánchez, M., de la Escosura-Muñiz, A. & Merkoçi, A. Improving sensitivity of gold nanoparticle-based lateral flow assays by using wax-printed pillars as delay barriers of microfluidics. Lab Chip 14, 4406–4414 (2014).

    CAS  PubMed  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Arben Merkoçi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Table 1.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing