Microchimica Acta

, Volume 181, Issue 1–2, pp 223–230 | Cite as

Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles

  • Suwussa Bamrungsap
  • Chayachon Apiwat
  • Warangkana Chantima
  • Tararaj Dharakul
  • Natpapas WiriyachaipornEmail author
Original Paper


We report on a lateral flow immunoassay (LFIA) for influenza A antigen using fluorescently-doped silica nanoparticles as reporters. The method is taking advantage of the high brightness and photostability of silica nanoparticles (doped with the dye Cy5) and the simplicity and rapidity of LFIA. The nucleoprotein of influenza A virion (one of its most abundant structural proteins) was used as a model to demonstrate a performance of the LFIA. Under optimized conditions and by using a portable strip reader, the fluorescence-based LFIA is capable of detecting a recombinant nucleoprotein as low as 250 ng · mL-1 using a sample volume of 100 μL, within 30 min, and without interference by other proteins. The successful detection of the nucleoprotein in infected allantoic fluid demonstrated the functionality of the method. By comparison with a commercial influenza A test based on gold nanoparticles as reporters, the system provides an 8-fold better sensitivity.


A rapid and sensitive lateral flow immunoassay for influenza A antigen was developed using fluorescently-doped silica nanoparticles. A sample containing nucleoprotein as a target analyte induced an accumulation of the fluorescent conjugates at the test spot. The signal was then measured quantitatively using a portable strip reader.


Fluorescence-doped silica nanoparticles Influenza A antigen detection Lateral flow immunoassay Biosensor 



This work was supported by grant from NANOTEC, Thailand.

Supplementary material

604_2013_1106_MOESM1_ESM.pdf (161 kb)
ESM 1 (PDF 161 kb)


  1. 1.
    Porstmann T, Kiessig ST (1992) Enzyme immunoassay techniques: an overview. J Immunol Methods 24:5–21CrossRefGoogle Scholar
  2. 2.
    Kurita R, Arai K, Nakamoto K, Kato D, Niwa O (2010) Development of electrogenerated chemiluminescence-based enzyme linked immunosorbent assay for sub-pM detection. Anal Chem 82:1692–1697CrossRefGoogle Scholar
  3. 3.
    Wingren C, Borrebaeck CA (2009) Antibody microarrays: current status and key technologies advances. OMICS 10:1692–1697Google Scholar
  4. 4.
    Wang Z, Lee J, Cossins AR, Brust M (2005) Microarray-based detection of protein binding and functionality by gold nanoparticle probes. Anal Chem 77:5770–5774CrossRefGoogle Scholar
  5. 5.
    Meyer MHF, Hartmann M, Keusgen M (2006) SPR-based immunosensor for the CRP detection-A new method to detect a well known protein. Biosens Bioelectron 21:1987–1990CrossRefGoogle Scholar
  6. 6.
    Temur E, Zengin A, Boyaci IH, Dudak FC, Torul H, Tamer U (2012) Attomole sensitivity of staphylococcal enterotoxin B detection using an aptamer-modified surface-enhanced Raman scattering probe. Anal Chem 84:10600–10606CrossRefGoogle Scholar
  7. 7.
    Yonzon CR, Haynes CL, Zhang X, Walsh JT, Van Duyne RP (2004) A glucose biosensor based on surface-enhanced Raman scattering: improved partition layer, temporal stability, reversibility, and resistance to serum protein interference. Anal Chem 76:78–85CrossRefGoogle Scholar
  8. 8.
    Posthuma-Trumpie GA, Korf J, van Amerogen A (2009) Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats: a literature survey. Anal Bioanal Chem 393:569–582CrossRefGoogle Scholar
  9. 9.
    Xu H, Mao X, Zeng Q, Wang S, Kawde AN, Liu G (2009) Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. Anal Chem 81:669–675CrossRefGoogle Scholar
  10. 10.
    Mizuike R, Sasaki T, Baba K, Iwamoto H, Shibai Y, Kosaka M, Kubota-Koketsu R, Yang CS, Du A, Sakudo A, Tsujikawa M, Yunoki M, Ikuta K (2011) Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1. Clin Vaccine Immunol 18:494–499CrossRefGoogle Scholar
  11. 11.
    Wiriyachaiporn S, Howarth PH, Bruce KD, Dailey LA (2013) Evaluation of a rapid lateral flow immunoassay for Staphylococcus aureus detection in respiratory samples. Diagn Microbiol Infect Dis 75:28–36CrossRefGoogle Scholar
  12. 12.
    Paek SH, Lee SH, Cho JH, Kim YS (2000) Development of rapid one-step immunochromatographic assay. Methods 22:53–60CrossRefGoogle Scholar
  13. 13.
    Choi DH, Lee SK, Oh YK, Bae BW, Lee SD, Kim S, Shin YB, Kim MG (2010) A dual gold nanoparticle conjugate-based lateral flow assay (LFA) method for the analysis of troponin I. Biosens Bioelectron 15:1999–2002CrossRefGoogle Scholar
  14. 14.
    Khreich N, Lamourette P, Boutal H, Devilliers K, Créminon C, Volland H (2008) Detection of Staphylococcus enterotoxin B using fluorescent immunoliposomes as label for immunochromatograhic testing. Anal Biochem 377:182–188CrossRefGoogle Scholar
  15. 15.
    O’Keeffe M, Crabbe P, Salden M, Wichers J, Van Peteghem C, Kohen F, Pieraccini G, Moneti G (2003) Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine. J Immunol Methods 278:117–126CrossRefGoogle Scholar
  16. 16.
    Kim YM, Oh SW, Jeong SY, Pyo DJ, Choi EY (2003) Development of an ultrarapid one-step fluorescence immunochromatographic assay system for the quantification of microcystins. Environ Sci Technol 37:1899–1904CrossRefGoogle Scholar
  17. 17.
    Ahna JS, Choia S, Janga SH, Changb HJ, Kimc JH, Nahmc KB, Oha SW, Choi EY (2003) Development of a point-of-care assay system for high-sensitivity C-reactive protein in whole blood. Clin Chim Acta 332:51–59CrossRefGoogle Scholar
  18. 18.
    Bae SW, Tan W, Hong JI (2012) Fluorescent dye-doped silica nanoparticles: new tools for bioapplications. Chem Commun 48:2270–2282CrossRefGoogle Scholar
  19. 19.
    Fare TL, Coffey EM, Dai HY, He YDD, Kessler DA, Kilian KA, Koch JE, LeProust E, Marton MJ, Meyer M, Stoughton RB, Tokiwa GY, Wang YQ (2003) Effect of atmospheric ozone on microarray data quality. Anal Chem 75:4672–4675CrossRefGoogle Scholar
  20. 20.
    He X, Chen J, Wang K, Qin D, Tan W (2007) Preparation of luminescent Cy5 doped core-shell SFNPs and its application as near-infrared fluorescent marker. Talanta 72:1519–1526CrossRefGoogle Scholar
  21. 21.
    Brigg MS, Burns DD, Cooper ME, Gregory SJ (2000) A pH sensitive fluorescent cyanine dye for biological applications. Chem Commun 23:2323–2324CrossRefGoogle Scholar
  22. 22.
    Tan W, Wang K, He X, Zhao XJ, Drake T, Wang L, Bagwe RP (2004) Bionanotechnology based on silica nanoparticles. Med Res Rev 24:621–638CrossRefGoogle Scholar
  23. 23.
    Santra S, Zhang P, Wang K, Tapec R, Tan W (2001) Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal Chem 73:4988–4993CrossRefGoogle Scholar
  24. 24.
    Smith JE, Medley CD, Tang Z, Shangguan D, Lofton C, Tan W (2007) Aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells. Anal Chem 79:3075–3082CrossRefGoogle Scholar
  25. 25.
    Watcharatanyatip K, Boonmoh S, Chaichoun K, Songserm T, Woratanti M, Dharakul T (2010) Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA. J Virol Methods 163:238–243CrossRefGoogle Scholar
  26. 26.
    World Health Organization (2002) WHO manual on animal influenza diagnosis and surveillance. Accessed 10 April 2013
  27. 27.
    King DJ (1991) Evaluation of different methods of inactivation of newcastle disease virus and avian influenza virus in egg fluids and serum. Avian Dis 35:505–514CrossRefGoogle Scholar
  28. 28.
    Barry RD, Davies P (1968) The sedimentation of influenza virus and its RNA in sucrose density gradients. J Gen Virol 2:59–69CrossRefGoogle Scholar
  29. 29.
    Stober W, Fink A, Bohn E (1968) Controlled growth of monodisperse silica spheres in the micron size range. J Colloid Interface Sci 26:62–69CrossRefGoogle Scholar
  30. 30.
    Wang L, Tan W (2006) Multicolor FRET silica nanoparticles by single wavelength excitation. Nano Lett 6:84–88CrossRefGoogle Scholar
  31. 31.
    Estévez MC, O’Donoghue MB, Chen X, Tan W (2009) Highly fluorescent dye-doped silica nanoparticles increase flow cytometry sensitivity for cancer cell monitoring. Nano Res 2:448–461CrossRefGoogle Scholar
  32. 32.
    Medley CD, Bamrungsap S, Tan W, Smith JE (2011) Aptamer-conjugated nanoparticles for cancer cell detection. Anal Chem 83:727–734CrossRefGoogle Scholar
  33. 33.
    Jian-umpunkul P, Thepthai C, Apiwat N, Chantima W, Poomputsa K, Wiriyachaiporn N, Dharakul T (2012) Improved sensitivity of influenza A antigen detection using a combined NP, M, and NS1 sandwich ELISA. J Virol Methods 185:24–31CrossRefGoogle Scholar
  34. 34.
    Farris LR, Wu N, Wang W, Clarizia LA, Wang X, McDonald MJ (2010) Immuno-interferometric sensor for the detection of influenza A nucleoprotein. Anal Bioanal Chem 396:667–674CrossRefGoogle Scholar
  35. 35.
    Ohtsuka K, Endo H, Morimoto K, Vuong BN, Ogawa H, Imai K, Takenaka S (2008) Detection of an antibody to avian influenza virus by an electrochemical immunoassay (eELISA). Anal Sci 24:1619–1622CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Suwussa Bamrungsap
    • 1
  • Chayachon Apiwat
    • 1
  • Warangkana Chantima
    • 2
  • Tararaj Dharakul
    • 1
    • 2
  • Natpapas Wiriyachaiporn
    • 1
    Email author
  1. 1.National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA)PathumthaniThailand
  2. 2.Department of Immunology, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand

Personalised recommendations