Microchimica Acta

, 185:210 | Cite as

Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine

  • Shuaijuan Han
  • Tianjiao Zhou
  • Bingjie Yin
  • Pingli He
Original Paper


The work describes a gold nanoparticle-based colorimetric enzyme-linked immunosorbent assay (ELISA) for ractopamine. The ELISA is based on an indirect competitive approach. In the presence of ractopamine, gold(III) ions are oxidized by H2O2 to form red AuNPs. On the other hand, the AuNP in solution are purple-blue due to aggregation if the sample does not contain ractopamine. The absorption, best measured at 560 nm, increases linearly in the 2 to 512 ng·mL−1 ractopamine concentration range, and the detection limit is as low as 0.35 ng·mL−1 in urine. Ractopamine can also be detected visually, even in the presence of other β-agonists and antibiotics. The results obtained by this method are consistent with those obtained by LC-MS/MS as demonstrated by analysis of sheep urine. The ELISA method described here is inexpensive, easy-to-use, and suitable for rapid screening of ractopamine in animal samples.

Graphical abstract

Schematic presentation of a colorimetric indirect competitive immunoassay for ractopamine. It is based on the use of catalase labeled IgG and the measurement of the absorption of red gold nanoparticles (AuNPs) that are generated by the reaction of gold ions with H2O2. In the absence of ractopamine, the solution becomes blue.


β-Agonist Colorimetric assay Nanogold Visual test Catalase Plasma-assisted method Sheep urine Immunoassay Enzyme-linked immunosorbent assay 



The Natural Science Foundation of China (31472126) is gratefully acknowledged.

Compliance with ethical standards

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

Supplementary material

604_2018_2736_MOESM1_ESM.doc (452 kb)
ESM 1 (DOC 451 kb)


  1. 1.
    European Food Safety Authority (2009) Safety evaluation of ractopamine. EFSA J 1041:1–52Google Scholar
  2. 2.
    He P, Zhang L, Yang T (2008) Determination of ractopamine in awine feed and urine using an indirect competitive immunoassay. J Anim Vet Adv 7(3):274–281Google Scholar
  3. 3.
    Kuiper HA, Noordam MY, van Dooren-Flipsen M, Schilt R, Roos AH (1998) Illegal use of beta-adrenergic agonists: European community. J Anim Sci 76(1):195–207CrossRefGoogle Scholar
  4. 4.
    Amelin VG, Korolev DS, Tret’Yakov AV (2015) Quechers sample preparation in the simultaneous determination of diethylstilbestrol and ractopamine in food by gas-liquid chromatography. J Anal Chem 70(4):419–423CrossRefGoogle Scholar
  5. 5.
    Turberg MP, Macy TD, Lewis JJ, Coleman MR (1995) Determination of ractopamine hydrochloride in swine and turkey tissues by liquid chromatography with coulometric detection. J AOAC Int 78(6):1394–1402Google Scholar
  6. 6.
    Li T, Cao J, Li Z, Wang X, He P (2016) Broad screening and identification of β-agonists in feed and animal body fluid and tissues using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry combined with spectra library search. Food Chem 192:188–196CrossRefGoogle Scholar
  7. 7.
    Gressler V, Franzen ARL, de Lima GJMM, Tavernari FC, Dalla Costa OA, Feddern V (2016) Development of a readily applied method to quantify ractopamine residue in meat and bone meal by Quechers-LC-MS/MS. J Chromatogr B 1015:192–200CrossRefGoogle Scholar
  8. 8.
    Wang P, Liu X, Su X, Zhu R (2015) Sensitive detection of beta-agonists in pork tissue with novel molecularly imprinted polymer extraction followed liquid chromatography coupled tandem mass spectrometry detection. Food Chem 184:72–79CrossRefGoogle Scholar
  9. 9.
    Elliott CT, Thompson CS, Arts C, Crooks S, van Baak MJ, Verheij ER, Baxter GA (1998) Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist. Analyst 123(5):1103–1107CrossRefGoogle Scholar
  10. 10.
    Shelver WL, Smith DJ (2000) Development of an immunoassay for the beta-adrenergic agonist ractopamine. J Immunoass 21(1):1–23CrossRefGoogle Scholar
  11. 11.
    Han S, Zhou T, Yin B, He P (2016) A sensitive and semi-quantitative method for determination of multi-drug residues in animal body fluids using multiplex dipstick immunoassay. Anal Chim Acta 927:64–71CrossRefGoogle Scholar
  12. 12.
    Shelver WL, Smith DJ (2003) Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis: comparative study with HPLC and ELISA. J Agric Food Chem 51(13):3715–3721CrossRefGoogle Scholar
  13. 13.
    Liu A, Lin J, Dai M, Ma B, Wu Y, Fang J, Zhang M (2016) Development of a monoclonal antibody-based Immunochromatographic assay detecting ractopamine residues in swine urine. Food Anal Methods 9(7):2016–2025CrossRefGoogle Scholar
  14. 14.
    Ma M, Zhu P, Pi F, Ji J, Sun X (2016) A disposable molecularly imprinted electrochemical sensor based on screen-printed electrode modified with ordered mesoporous carbon and gold nanoparticles for determination of ractopamine. J Electroanal Chem 775:171–178CrossRefGoogle Scholar
  15. 15.
    Shen L, Li Z, He P (2010) Electrochemical behavior of β 2-agonists at graphite nanosheet modified electrodes. Electrochem Commun 12(7):876–881CrossRefGoogle Scholar
  16. 16.
    Wei Q, Wang Q, Wang H, Gu H, Zhang Q, Gao X, Qi B (2015) Formation of flowerlike gold nanostructure on ordered mesoporous carbon electrode and its application in electrochemical determination of ractopamine. Mater Lett 147:58–60CrossRefGoogle Scholar
  17. 17.
    Zhou Y, Wang P, Su X, Zhao H, He Y (2014) Sensitive immunoassay for the β-agonist ractopamine based on glassy carbon electrode modified with gold nanoparticles and multi-walled carbon nanotubes in a film of poly-arginine. Microchim Acta 181(15–16):1973–1979CrossRefGoogle Scholar
  18. 18.
    Scampicchio M, Arecchi A, Mannino S (2009) Optical nanoprobes based on gold nanoparticles for sugar sensing. Nanotechnology 20(13):135501CrossRefGoogle Scholar
  19. 19.
    He P, Shen L, Liu R, Luo Z, Li Z (2011) Direct detection of beta-agonists by use of gold nanoparticle-based colorimetric assays. Anal Chem 83(18):6988–6995CrossRefGoogle Scholar
  20. 20.
    Shen L, Chen J, Li N, He P, Li Z (2014) Rapid colorimetric sensing of tetracycline antibiotics with in situ growth of gold nanoparticles. Anal Chim Acta 839:83–90CrossRefGoogle Scholar
  21. 21.
    Zhang Y, Ma H, Wu D, Li Y, Du B, Wei Q (2015) Label-free immunosensor based on au@ag 2 S nanoparticles/magnetic chitosan matrix for sensitive determination of ractopamine. J Electroanal Chem 741:14–19CrossRefGoogle Scholar
  22. 22.
    Wang P, Su X, Shi L, Yuan Y (2016) An aptamer based assay for the β-adrenergic agonist ractopamine based on aggregation of gold nanoparticles in combination with a molecularly imprinted polymer. Microchim Acta 183(11):2899–2905CrossRefGoogle Scholar
  23. 23.
    Luo Y, Liu X, Guo J, Gao H, Li Y, Xu J, Shen F, Sun C (2016) Visual screening and colorimetric determination of clenbuterol and ractopamine using unmodified gold nanoparticles as probe. J Nanosci Nanotechnol 16(1):548–554CrossRefGoogle Scholar
  24. 24.
    de la Rica R, Stevens MM (2012) Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat Nanotechnol 7(12):821–824CrossRefGoogle Scholar
  25. 25.
    Cecchin D, de la Rica R, Bain RES, Finnis MW, Stevens MM, Battaglia G (2014) Plasmonic ELISA for the detection of gp120 at ultralow concentrations with the naked eye. Nano 6(16):9559–9562Google Scholar
  26. 26.
    Gobbo P, Biondi MJ, Feld JJ, Workentin MS (2013) Arresting the time-dependent H2O2 mediated synthesis of gold nanoparticles for analytical detection and preparative chemistry. J Mater Chem B 1(33):4048–4051CrossRefGoogle Scholar
  27. 27.
    Li S, He P (2007) An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine. Electrochem Commun 9:657–662CrossRefGoogle Scholar
  28. 28.
    Bouyahia N, Hamlaoui ML, Hnaien M, Lagarde F, Jaffrezic-Renault N (2011) Impedance spectroscopy and conductometric biosensing for probing catalase reaction with cyanide as ligand and inhibitor. Bioelectrochemistry 80(2):155–161CrossRefGoogle Scholar
  29. 29.
    Gu H, Liu L, Song S, Kung H, Xu C (2016) Development of an immunochromatographic strip assay for ractopamine detection using an ultrasensitive monoclonal antibody. Food Agric Immunol 27:471–483CrossRefGoogle Scholar
  30. 30.
    Zhu C, Zhang G, Huang Y, Yan J, Chen A (2017) Aptamer based ultrasensitive determination of the β-adrenergic agonist ractopamine using PicoGreen as a fluorescent DNA probe. Microchim Acta 184(2):439–444CrossRefGoogle Scholar
  31. 31.
    Liu H, Fang G, Wang S (2014) Molecularly imprinted optosensing material based on hydrophobic CdSe quantum dots via a reverse microemulsion for specific recognition of ractopamine. Biosens Bioelectron 55:127–132CrossRefGoogle Scholar
  32. 32.
    Chen S, Zhang J, Gan N, Hu F, Li T, Cao Y, Pan D (2015) An on-site immunosensor for ractopamine based on a personal glucose meter and using magnetic β-cyclodextrin-coated nanoparticles for enrichment, and an invertase-labeled nanogold probe for signal amplification. Microchim Acta 182(3–4):815–822CrossRefGoogle Scholar
  33. 33.
    Yao T, Gu X, Li T, Li J, Li J, Zhao Z, Wang J, Qin Y, She Y (2016) Enhancement of surface plasmon resonance signals using a MIP/GNPs/rGO nano-hybrid film for the rapid detection of ractopamine. Biosens Bioelectron 75:96–100CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Animal Nutrition, College of Animal Science and TechnologyChina Agricultural UniversityBeijingPeople’s Republic of China

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