Microchimica Acta

, Volume 184, Issue 10, pp 4159–4165 | Cite as

Rapid single-step upconversion-linked immunosorbent assay for diclofenac

  • Antonín Hlaváček
  • Miroslav Peterek
  • Zdeněk Farka
  • Matthias J. Mickert
  • Leonhard Prechtl
  • Dietmar Knopp
  • Hans H. GorrisEmail author
Original Paper


The non-steroidal anti-inflammatory drug and analgesic diclofenac is a common micropollutant in water. A direct competitive upconversion-linked immunosorbent assay (ULISA) for diclofenac has been developed that is based on a nanoparticulate tracer consisting of a NaYF4:Yb,Er core (diameter of ~90 nm, excitation and emission wavelenghts 980 and 535 nm, respectively) enclosed by a carboxylated silica shell and finally coated with diclofenac-conjugated bovine γ-globulin. The proteinaceous coating prevents non-specific adsorption of the tracer to the microtiter plate and provides a structurally flexible linker for surface-exposed diclofenac to warrant efficient competition with free (analyte) diclofenac in real water samples. The tracer was purified by gel electrophoresis and lyophilized. Both processes have no adverse effects on the immunoassay. All assay components can be stored in a dry state without a cooling chain, and can be reactivated on demand. Hence, this ULISA is well suited for environmental monitoring in low-resource settings. The ULISA has a similar limit of detection (20 pg mL−1; equivalent to 70 pM) as the conventional ELISA, but the time for analysis is reduced to 70 min because no enzymatic amplification steps are involved.

Graphical abstract

Photon-upconversion nanoparticles (UCNP) doped with Yb3+ and Er3+ emit visible light under 980-nm excitation. A luminescent tracer consisting of a UCNP covered by diclofenac-modified bovine γ-globulin allows for the direct competitive detection of diclofenac in a single-step immunoassay without optical background interference


Bioconjugation Electrophoretic purification Immunoassay Luminescence Pharmaceutical micropollutant Photon-upconversion 



We acknowledge financial support from the COST Action CM1403 “The European Upconversion Network: From the Design of Photon-Upconverting Nanomaterials to Biomedical Applications”. H.H.G. acknowledges funding from the German Research Foundation for a Heisenberg Fellowship (DFG, Grant GO 1968/5-1 and GO 1968/6-1). Further funding was provided by the Czech Ministry of Education, Youth and Sports (COST CZ project LD15023), ANR-DFG program (project NArBioS, Grant no. ANR-11-INTB-1013) and the Grant Agency of the Czech Republic (P20612G014 GACR). CIISB research infrastructure project LM2015043 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography.

Compliance with ethical standards

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

Supplementary material

604_2017_2456_MOESM1_ESM.docx (2.1 mb)
ESM 1 (DOCX 2177 kb)


  1. 1.
    Wang L, Wang KM, Santra S, Zhao XJ, Hilliard LR, Smith JE, Wu JR, Tan WH (2006) Watching silica nanoparticles glow in the biological world. Anal Chem 78:646–654. doi: 10.1021/ac0693619 CrossRefGoogle Scholar
  2. 2.
    Jans H, Huo Q (2012) Gold nanoparticle-enabled biological and chemical detection and analysis. Chem Soc Rev 41:2849–2866. doi: 10.1039/c1cs15280g CrossRefGoogle Scholar
  3. 3.
    Osterfeld SJ, Yu H, Gaster RS, Caramuta S, Xu L, Han SJ, Hall DA, Wilson RJ, Sun SH, White RL, Davis RW, Pourmand N, Wang SX (2008) Multiplex protein assays based on real-time magnetic nanotag sensing. Proc Natl Acad Sci U S A 105:20637–20640. doi: 10.1073/pnas.0810822105 CrossRefGoogle Scholar
  4. 4.
    Čunderlová V, Hlaváček A, Horňáková V, Peterek M, Němeček D, Hampl A, Eyer L, Skládal P (2016) Catalytic nanocrystalline coordination polymers as an efficient peroxidase mimic for labeling and optical immunoassays. Microchim Acta 183:651–658. doi: 10.1007/s00604-015-1697-z CrossRefGoogle Scholar
  5. 5.
    Hlaváček A, Bouchal P, Skládal P (2012) Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification. Microchim Acta 176:287–293. doi: 10.1007/s00604-011-0729-6 CrossRefGoogle Scholar
  6. 6.
    Hlaváček A, Farka Z, Hübner M, Horňáková V, Němeček D, Niessner R, Skládal P, Knopp D, Gorris HH (2016) Competitive upconversion-linked immunosorbent assay for the sensitive detection of diclofenac. Anal Chem 88:6011–6017. doi: 10.1021/acs.analchem.6b01083 CrossRefGoogle Scholar
  7. 7.
    Päkkilä H, Ylihärsilä M, Lahtinen S, Hattara L, Salminen N, Arppe R, Lastusaari M, Saviranta P, Soukka T (2012) Quantitative multianalyte microarray immunoassay utilizing upconverting phosphor technology. Anal Chem 84:8628–8634. doi: 10.1021/ac301719p CrossRefGoogle Scholar
  8. 8.
    Achatz DE, Ali R, Wolfbeis OS (2011) Luminescent chemical sensing, biosensing, and screening using upconverting nanoparticles. Top Curr Chem 300:29–50. doi: 10.1007/128_2010_98 CrossRefGoogle Scholar
  9. 9.
    Auzel F (2004) Upconversion and anti-stokes processes with f and d ions in solids. Chem Rev 104:139–173. doi: 10.1021/cr020357g CrossRefGoogle Scholar
  10. 10.
    Haase M, Schäfer H (2011) Upconverting nanoparticles. Angew Chem Int Ed 50:5808–5829. doi: 10.1002/anie.201005159 CrossRefGoogle Scholar
  11. 11.
    Yang Y (2014) Upconversion nanophosphors for use in bioimaging, therapy, drug delivery and bioassays. Microchim Acta 181:263–294. doi: 10.1007/s00604-013-1139-8 CrossRefGoogle Scholar
  12. 12.
    Gorris HH, Ali R, Saleh SM, Wolfbeis OS (2011) Tuning the dual emission of photon-upconverting nanoparticles for ratiometric multiplexed encoding. Adv Mater 23:1652–1655. doi: 10.1002/adma.201004697 CrossRefGoogle Scholar
  13. 13.
    Ternes TA (2001) Analytical methods for the determination of pharmaceuticals in aqueous environmental samples. Trac-Trend Anal Chem 20:419–434. doi: 10.1016/S0165-9936(01)00078-4 CrossRefGoogle Scholar
  14. 14.
    Perez S, Barcelo D (2007) Application of advanced MS techniques to analysis and identification of human and microbial metabolites of pharmaceuticals in the aquatic environment. Trac-Trend Anal Chem 26:494–514. doi: 10.1016/j.trac.2007.05.004 CrossRefGoogle Scholar
  15. 15.
    Heberer T (2002) Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol Lett 131:5–17. doi: 10.1016/S0378-4274(02)00041-3 CrossRefGoogle Scholar
  16. 16.
    Koutsouba V, Heberer T, Fuhrmann B, Schmidt-Baumler K, Tsipi D, Hiskia A (2003) Determination of polar pharmaceuticals in sewage water of Greece by gas chromatography-mass spectrometry. Chemosphere 51:69–75. doi: 10.1016/S0045-6535(02)00819-6 CrossRefGoogle Scholar
  17. 17.
    Oaks JL, Gilbert M, Virani MZ, Watson RT, Meteyer CU, Rideout BA, Shivaprasad HL, Ahmed S, Chaudhry MJI, Arshad M, Mahmood S, Ali A, Khan AA (2004) Diclofenac residues as the cause of vulture population decline in Pakistan. Nature 427:630–633. doi: 10.1038/nature02317 CrossRefGoogle Scholar
  18. 18.
    Petrovic M, Hernando MD, Diaz-Cruz MS, Barcelo D (2005) Liquid chromatography-tandem mass spectrometry for the analysis of pharmaceutical residues in environmental samples: a review. J Chromatogr A 1067:1–14. doi: 10.1016/j.chroma.2004.10.110 CrossRefGoogle Scholar
  19. 19.
    Deng AP, Himmelsbach M, Zhu QZ, Frey S, Sengl M, Buchberger W, Niessner R, Knopp D (2003) Residue analysis of the pharmaceutical diclofenac in different water types using ELISA and GC-MS. Environ Sci Technol 37:3422–3429. doi: 10.1021/es0341945 CrossRefGoogle Scholar
  20. 20.
    Huebner M, Weber E, Niessner R, Boujday S, Knopp D (2015) Rapid analysis of diclofenac in freshwater and wastewater by a monoclonal antibody-based highly sensitive ELISA. Anal Bioanal Chem 407:8873–8882. doi: 10.1007/s00216-015-9048-9 CrossRefGoogle Scholar
  21. 21.
    Sedlmeier A, Gorris HH (2015) Surface modification and characterization of photon-upconverting nanoparticles for bioanalytical applications. Chem Soc Rev 44:1526–1560. doi: 10.1039/c4cs00186a CrossRefGoogle Scholar
  22. 22.
    Hlaváček A, Sedlmeier A, Skládal P, Gorris HH (2014) Electrophoretic characterization and purification of silica-coated photon-upconverting nanoparticles and their bioconjugates. ACS Appl Mater Interfaces 6:6930–6935. doi: 10.1021/am500732y CrossRefGoogle Scholar
  23. 23.
    Petrie B, Barden R, Kasprzyk-Hordern B (2015) A review on emerging contaminants in wastewaters and the environment: current knowledge, understudied areas and recommendations for future monitoring. Water Res 72:3–27. doi: 10.1010/j.watres.2014.08.053 CrossRefGoogle Scholar
  24. 24.
    Osório MV, Reis S, Lima JLFC, Segundo MA (2017) Analytical features of diclofenac evaluation in water as a potential marker for anthriopogenic pollution. Curr Pharm Anal 13:39–47. doi: 10.2174/1573412912666160422123841 CrossRefGoogle Scholar
  25. 25.
    Wang F, Han Y, Lim CS, Lu YH, Wang J, Xu J, Chen HY, Zhang C, Hong MH, Liu XG (2010) Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping. Nature 463:1061–1065. doi: 10.1038/nature08777 CrossRefGoogle Scholar
  26. 26.
    Fuller BJ (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo-Lett 25:375–388Google Scholar
  27. 27.
    Sedlmeier A, Hlaváček A, Birner L, Mickert MJ, Muhr V, Hirsch T, Corstjens PL, Tanke HJ, Soukka T, Gorris HH (2016) Highly sensitive laser scanning of photon-upconverting nanoparticles on a macroscopic scale. Anal Chem 88:1835–1841. doi: 10.1021/acs.analchem.5b04147 CrossRefGoogle Scholar
  28. 28.
    Al-Odaini NA, Zakaria MP, Yaziz MI, Surif S (2010) Multi-residue analytical method for human pharmaceuticals and synthetic hormones in river water and sewage effluents by solid-phase extraction and liquid chromatography-tandem mass spectrometry. J Chromatogr A 1217:6791–6806. doi: 10.1016/j.chroma.2010.08.033 CrossRefGoogle Scholar
  29. 29.
    Altintas Z, Guerreiro A, Piletsky SA, Tothill IE (2015) NanoMIP based optical sensor for pharmaceuticals monitoring. Sensors Actuators B Chem 213:305–313. doi: 10.1016/j.snb.2015.02.043 CrossRefGoogle Scholar
  30. 30.
    Shiravand T, Azadbakht A (2017) Impedimetric biosensor based on bimetallic AgPt nanoparticle-decorated carbon nanotubes as highly conductive film surface. J Solid State Electrochem 21:1699–1711. doi: 10.1007/s10008-017-3532-4
  31. 31.
    Azadbakht A, Beirnvand S (2017) Voltammetric aptamer-based switch probes for sensing diclofenac using a glassy carbon electrode modified with a composite prepared from gold nanoparticles, carbon nanotubes and amino-functionalized Fe3O4 nanoparticles. Microchim Acta 184:2825–2835. doi: 10.1007/s00604-017-2285-1
  32. 32.
    Guitart C, Readman JW (2010) Critical evaluation of the determination of pharmaceuticals, personal care products, phenolic endocrine disrupters and faecal steroids by GC/MS and PTV-GC/MS in environmental waters. Anal Chim Acta 658:32–40. doi: 10.1016/j.aca.2009.10.066 CrossRefGoogle Scholar
  33. 33.
    Ahrer W, Scherwenk E, Buchberger W (2001) Determination of drug residues in water by the combination of liquid chromatography or capillary electrophoresis with electrospray mass spectrometry. J Chromatogr A 910:69–78. doi: 10.1016/S0021-9673(00)01187-0 CrossRefGoogle Scholar
  34. 34.
    Malá Z, Gebauer P, Boček P (2016) Capillary isotachophoresis with ESI-MS detection: methodology for highly sensitive analysis of ibuprofen and diclofenac in waters. Anal Chim Acta 907:1–6. doi: 10.1016/j.aca.2015.11.040 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria 2017

Authors and Affiliations

  • Antonín Hlaváček
    • 1
    • 2
    • 3
  • Miroslav Peterek
    • 2
  • Zdeněk Farka
    • 1
    • 2
  • Matthias J. Mickert
    • 1
  • Leonhard Prechtl
    • 4
  • Dietmar Knopp
    • 4
  • Hans H. Gorris
    • 1
    Email author
  1. 1.Institute of Analytical Chemistry, Chemo- and BiosensorsUniversity of RegensburgRegensburgGermany
  2. 2.CEITEC - Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic
  3. 3.Institute of Analytical Chemistry of the Czech Academy of Siences602 00 BrnoCzech Republic
  4. 4.Institute of HydrochemistryTechnical University of MunichMunichGermany

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