Microchimica Acta

, 186:429 | Cite as

Fluorescent silver nanoclusters as antibody label in a competitive immunoassay for the complement factor H

  • Eva Valencia
  • María Cruz-Alonso
  • Lydia Álvarez
  • Héctor González-IglesiasEmail author
  • Beatriz FernándezEmail author
  • Rosario Pereiro
Original Paper


Silver nanoclusters (AgNCs) were investigated as labels for the development of a fluoroimmunoassay for the complement factor H (CFH). The reductive one-pot synthesis of AgNCs using lipoic acid as a ligand was optimized by varying the concentration of NaBH4, the temperature and the reaction time. The average diameter and crystal structure of the AgNCs (which display red fluorescence) were determined by HR-TEM. The silver concentration was quantified by ICP-MS. Labelling of the antibody against CFH with AgNCs was optimized. The antibody was labeled with the AgNCs without compromising the recognition capabilities of the antibody. A competitive fluoroimmunoassay was then developed. Fluorescence is measured at excitation/emission maxima of 430/660 nm. The assay has a 0.4 ng mL−1 detection limit and a linear range that extends from 1.2 to 23 ng mL−1. The results compare favorably with those obtained by a commercial ELISA kit. The method was applied to the determination of CFH in spiked human serum.

Graphical abstract

Schematic presentation of the method for the determination of complement factor H (CFH) protein in human blood by using a CFH-antibody labelled with fluorescent silver nanoclusters.


Fluoroimmunoassay Lipoic acid One-pot synthesis Carbodiimide Labelling Human serum 



This work was supported through project CTQ2016-79015-R by Agencia Estatal de Investigación (Spain) and FEDER. B. Fernandez acknowledges her contract RYC-2014-14985 to the Spanish Ministry of Economy and Competitiveness through the “Ramón y Cajal Program”. The Instituto Oftalmológico Fernández-Vega and Fundación de Investigación Oftalmológica acknowledge support from “Cátedra Rafael del Pino” and from Instituto de Desarrollo Económico del Principado de Asturias (IDEPA) and FEDER (project IDE/2016/000214).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3554_MOESM1_ESM.docx (369 kb)
ESM 1 (DOCX 369 kb)


  1. 1.
    Klein R, Peto T, Bird A, Vannewkirk MR (2004) The epidemiology of age-related macular degeneration. Am J Ophthalmol 137:486–495CrossRefGoogle Scholar
  2. 2.
    Lim LS, Mitchell P, Seddon JM, Holz FG, Wong TY (2012) Age-related macular degeneration. Lancet 379:1728–1738CrossRefGoogle Scholar
  3. 3.
    Crabb JW (2014) The proteomics of drusen. Cold Spring Harb Perspect Med 4:a017194CrossRefGoogle Scholar
  4. 4.
    Wang L, Clark ME, Crossman DK, Kojima K, Messinger JD, Mobley JA, Curcio CA (2010) Abundant lipid and protein components of drusen. PLoS One 5:e10329CrossRefGoogle Scholar
  5. 5.
    Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, Haynes C, Henning AK, San Giovanni JP, Mane SM, Mayne ST, Bracken MB, Ferris FL, Ott J, Barnstable C, Hoh J (2005) Complement factor H polymorphism in age related macular degeneration. Science 308:385–389CrossRefGoogle Scholar
  6. 6.
    Geerlings MJ, de Jong EK, den Hollander AI (2017) The complement system in age-related macular degeneration: a review of rare genetic variants and implications for personalized treatment. Mol Immunol 84:65–76CrossRefGoogle Scholar
  7. 7.
    Triebwasser MP, Roberson ED, Yu Y, Schramm EC, Wagner EK, Raychaudhuri S, Seddon JM, Atkinson JP (2015) Rare variants in the functional domains of complement factor H are associated with age-related macular degeneration. Invest Ophthalmol Vis Sci 56:6873–6878CrossRefGoogle Scholar
  8. 8.
    Geerlings MJ, Kremlitzka M, Bakker B, Nilsson SC, Saksens NT, Lechanteur YT, Pauper M, Corominas J, Fauser S, Hoyng CB, Blom AM, de Jong EK, den Hollander AI (2017) The functional effect of rare variants in complement genes on C3b degradation in patients with age-related macular degeneration. JAMA Ophthalmol 135:39–46CrossRefGoogle Scholar
  9. 9.
    Gemenetzi M, Lotery AJ (2016) Complement pathway biomarkers and age-related macular degeneration. Eye (Lond) 30:1–14CrossRefGoogle Scholar
  10. 10.
    Kim YH, He S, Kase S, Kitamura M, Ryan SJ, Hinton DR (2009) Regulated secretion of complement factor H by RPE and its role in RPE migration. Graefes Arch Clin Exp Ophthalmol 247:651–659CrossRefGoogle Scholar
  11. 11.
    Chen M, Forrester JV, Xu H (2007) Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments. Exp Eye Res 84:635–645CrossRefGoogle Scholar
  12. 12.
    Vellaisamy K, Li G, Ko CN, Zhong HJ, Fatima S, Kwan HY, Wong CY, Kwong WJ, Tan W, Leung CH, Ma DL (2018) Cell imaging of dopamine receptor using agonist labeling iridium(III) complex. Chem Sci 9:1119–1125CrossRefGoogle Scholar
  13. 13.
    Zhan JJ, Cheng FF, Li JJ, Zhu JJ, Lu Y (2016) Fluorescent nanoprobes for sensing and imaging of metal ions: recent advances and future perspectives. Nano Today 11:309–329CrossRefGoogle Scholar
  14. 14.
    Zhang L, Wang E (2014) Metal nanoclusters: new fluorescent probes for sensors and bioimaging. Nano Today 9:132–157CrossRefGoogle Scholar
  15. 15.
    Song X-R, Goswami N, Yang H-H, Xie J (2016) Functionalization of metal nanoclusters for biomedical applications. Analyst 141:3126–3140CrossRefGoogle Scholar
  16. 16.
    Chen L-Y, Wang C-W, Yuan Z, Chang H-T (2015) Fluorescent gold nanoclusters: recent advances in sensing and imaging. Anal Chem 87:216–229CrossRefGoogle Scholar
  17. 17.
    Alonso MC, Trapiella-Alfonso L, Fernández JMC, Pereiro R, Sanz-Medel A (2016) Functionalized gold nanoclusters as fluorescent labels for immunoassays: application to human serum immunoglobulin E determination. Biosens Bioelectron 77:1055–1061CrossRefGoogle Scholar
  18. 18.
    Xu H, Suslick KS (2010) Water-soluble fluorescent silver nanoclusters. Adv Mater 22:1078–1082CrossRefGoogle Scholar
  19. 19.
    Diez I, Ras RHA (2011) Fluorescent silver nanoclusters. Nanoscale 3:1963–1970CrossRefGoogle Scholar
  20. 20.
    Luo Z, Zheng K, Xie J (2014) Engineering ultrasmall water-soluble gold and silver nanoclusters for biomedical applications. Chem Commun 50:5143–5155CrossRefGoogle Scholar
  21. 21.
    Fernández-Ujados M, Trapiella-Alfonso L, Costa JM, Pereiro R, Sanz-Medel A (2013) One step aqueous synthesis of fluorescent copper nanoclusters by direct metal reduction. Nanotechnology 24:495601CrossRefGoogle Scholar
  22. 22.
    Chen D, Gao S, Ge W, Li Q, Jiang H, Wang X (2014) One-step rapid synthesis of fluorescent platinum nanoclusters for cellular imaging and photothermal treatment. RSC Adv 4:40141–40145CrossRefGoogle Scholar
  23. 23.
    Chen Y, Phipps ML, Werner JH, Chakraborty S, Martinez JS (2018) DNA templated metal nanoclusters: from emergent properties to unique applications. Acc Chem Res 51:2756–2763CrossRefGoogle Scholar
  24. 24.
    Yeh H-C, Sharma J, Han JJ, Martinez JS, Werner JH (2010) A DNA-silver nanocluster probe that fluoresces upon hybridization. Nano Lett 10:3106–3110CrossRefGoogle Scholar
  25. 25.
    Li B, Xu L, Chen Y, Zhu W, Shen X, Zhu C, Luo J, Li X, Hong J, Zhou X (2017) Sensitive and label-free fluorescent detection of transcription factors based on DNA-ag nanoclusters molecular beacons and exonuclease III-assisted signal amplification. Anal Chem 89:7316–7323CrossRefGoogle Scholar
  26. 26.
    Li J, Zhu J-J, Xu K (2014) Fluorescent metal nanoclusters: from synthesis to applications. Trends Analyt Chem 58:90–98CrossRefGoogle Scholar
  27. 27.
    Zheng K, Yuan X, Goswami N, Zhang Q, Xie J (2014) Recent advances in the synthesis, characterization, and biomedical applications of ultrasmall thiolated silver nanoclusters. RSC Adv 4:60581–60596CrossRefGoogle Scholar
  28. 28.
    Adhikari B, Banerjee A (2010) Facile synthesis of water-soluble fluorescent silver nanoclusters and HgII sensing. Chem Mater 22:4364–4371CrossRefGoogle Scholar
  29. 29.
    Shang L, Dörlich RM, Trouillet V, Bruns M, Nienhaus GU (2012) Ultrasmall fluorescent silver nanoclusters: protein adsorption and its effects on cellular responses. Nano Res 5:531–542CrossRefGoogle Scholar
  30. 30.
    Ren SH, Liu SG, Ling Y, Li NB, Luo HQ (2018) Fluorescence detection of melamine based on inhibiting Cu2+-induced disaggregation of red-emitting silver nanoclusters. Spectrochim Acta A 201:112–118CrossRefGoogle Scholar
  31. 31.
    Shu T, Lin X, Zhou Z, Zhao D, Xue F, Zeng F, Wang J, Wang C, Su L, Zhang X (2019) Understanding stimuli-responsive oligomer shell of silver nanoclusters with aggregation-induced emission via chemical etching and their use as sensors. Sensor Actuat B-Chem 286:198–205CrossRefGoogle Scholar
  32. 32.
    Rogers AB, Cormier KS, Fox JG (2006) Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry. Lab Investig 86:526–533CrossRefGoogle Scholar
  33. 33.
    Yu Y, Yao Q, Luo Z, Yuan X, Lee JY, Xie J (2013) Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters. Nanoscale 5:4606–4620CrossRefGoogle Scholar
  34. 34.
    Garcia MED, Sanz-Medel A (1986) Facile chemical deoxygenation of micellar solutions for room temperature phosphorescence. Anal Chem 58:1436–1440CrossRefGoogle Scholar
  35. 35.
    Trapiella-Alfonso L, Menéndez-Miranda M, Costa-Fernández JM, Pereiro R, Sanz-Medel A (2014) Nanostructural transformations of silver nanoclusters occurring during their synthesis and after interaction with UV-light. Mater Res Express 1:015039CrossRefGoogle Scholar
  36. 36.
    Sofat R, Mangione PP, Gallimore JR, Hakobyan S, Hughes TR, Shah T, Goodship T, D'Aiuto F, Langenberg C, Wareham N, Morgan BP, Pepys MB, Hingorani AD (2013) Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay. J Immunol Methods 390:63–73CrossRefGoogle Scholar
  37. 37.
    Hakobyan S, Harris CL, Tortajada A, Goicochea de Jorge E, García-Layana A, Fernández-Robredo P, Rodríguez de Córdoba S, Morgan BP (2008) Measurement of factor H variants in plasma using variant-specific monoclonal antibodies: application to assessing risk of age-related macular degeneration. Invest Ophthalmol Vis Sci 49:1983–1990CrossRefGoogle Scholar
  38. 38.
    Hakobyan S, Tortajada A, Harris CL, Rodríguez de Córdoba S, Morgan BP (2010) Variant-specific quantification of factor H in plasma reveals null alleles associated with a typical hemolytic uremic syndrome. Kidney Int 78:782–788CrossRefGoogle Scholar
  39. 39.
    Cho YM, Mizuta Y, Akagi J, Toyoda T, Sone M, Ogawa K (2018) Size-dependent acute toxicity of silver nanoparticles in mice. J Toxicol Pathol 31:73–80CrossRefGoogle Scholar
  40. 40.
    Zhang L, Goswami N, Xie J, Zhang B, He Y (2017) Unraveling the molecular mechanism of photosynthetic toxicity of highly fluorescent silver nanoclusters to Scenedesmus obliquus. Sci Rep 7:16432 (12pp)CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Physical and Analytical ChemistryUniversity of OviedoOviedoSpain
  2. 2.Instituto Universitario Fernández-Vega (Fundación de Investigación Oftalmológica, Universidad de Oviedo)OviedoSpain
  3. 3.Instituto Oftalmológico Fernández-VegaOviedoSpain

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