Concentration and preservation of very low abundance biomarkers in urine, such as human growth hormone (hGH), by Cibacron Blue F3G-A loaded hydrogel particles


Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels.


  1. [1]

    Gonzalez-Buitrago, J. M.; Ferreira, L.; Lorenzo, I. Urinary proteomics. Clin. Chim. Acta 2007, 375, 49–56.

    PubMed  Article  CAS  Google Scholar 

  2. [2]

    Barratt, J.; Topham, P. Urine proteomics: The present and future of measuring urinary protein components in disease. CMAJ 2007, 177, 361–368.

    PubMed  Google Scholar 

  3. [3]

    Pisitkun, T.; Johnstone, R.; Knepper, M. A. Discovery of urinary biomarkers. Mol. Cell. Proteomics 2006, 5, 1760–1771.

    PubMed  Article  CAS  Google Scholar 

  4. [4]

    Kreunin, P.; Zhao, J.; Rosser, C.; Urquidi, V.; Lubman, D. M.; Goodison, S. Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling. J. Proteome. Res. 2007, 6, 2631–2639.

    PubMed  Article  CAS  Google Scholar 

  5. [5]

    Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Nanomedicine: Current status and future prospects. Faseb. J. 2005, 19, 311–330.

    PubMed  Article  CAS  Google Scholar 

  6. [6]

    Jain, K. K. Applications of nanobiotechnology in clinical diagnostics. Clin. Chem. 2007, 53, 2002–2009.

    PubMed  Article  CAS  Google Scholar 

  7. [7]

    Tanaka, T.; Sato, E.; Hirokawa, Y.; Hirotsu, S.; Peetermans, J. Critical kinetics of volume phase transition of gels. Phys. Rev. Lett. 1985, 55, 2455.

    PubMed  Article  ADS  CAS  Google Scholar 

  8. [8]

    Jones, C. D.; Lyon, L. A. Synthesis and characterization of multiresponsive core-shell microgels. Macromolecules 2000, 33, 8301–8306.

    Article  CAS  Google Scholar 

  9. [9]

    Duracher, D.; Sauzedde, F.; Elaissari, A.; Perrin, A.; Pichot, C. Cationic amino-containing n-isopropylacrylamide-styrene copolymer latex particles: 1-Particle size and morphology vs polymerization process. Colloid. Polym. Sci. 1998, 276, 219–231.

    Article  CAS  Google Scholar 

  10. [10]

    Suzuki, A. and Tanaka, T. Phase transition in polymer gels induced by visible light. Nature 1990, 346, 345–347.

    Article  ADS  CAS  Google Scholar 

  11. [11]

    Tanaka, T.; Nishio, I.; Sun, S. -T.; Ueno-Nishio, S. Collapse of gels in an electric field. Science.1982, 218, 467–469.

    PubMed  Article  ADS  CAS  Google Scholar 

  12. [12]

    Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 2002, 54, 3–12.

    PubMed  Article  CAS  Google Scholar 

  13. [13]

    Liu, J.; Pelton, R.; Hrymak, A. N. Properties of poly (n-isopropylacrylamide)-grafted colloidal silica. J. Colloid Interf. Sci. 2000, 227, 408–411.

    Article  CAS  Google Scholar 

  14. [14]

    Cavalieri, F.; Chiessi, E.; Villa, R.; Vigano, L.; Zaffaroni, N.; Telling, M. F.; Paradossi, G. Novel PVA-based hydrogel microparticles for doxorubicin delivery. Biomacromolecules 2008, 9, 1967–1973.

    PubMed  Article  CAS  Google Scholar 

  15. [15]

    Zhang, X. Z.; Jo Lewis, P.; Chu, C. C. Fabrication and characterization of a smart drug delivery system: Microsphere in hydrogel. Biomaterials 2005, 26, 3299–3309.

    PubMed  Article  CAS  Google Scholar 

  16. [16]

    Luchini, A.; Geho, D. H.; Bishop, B.; Tran, D.; Xia, C.; Dufour, R. L.; Jones, C. D.; Espina, V.; Patanarut, A.; Zhou, W. et al. Smart hydrogel particles: Biomarker harvesting: One-step affinity purification, size exclusion, and protection against degradation. Nano Lett. 2008, 8, 350–361.

    PubMed  Article  CAS  Google Scholar 

  17. [17]

    Tuncel, A.; Ozdemir, A. Thermally reversible VPBANIPAM copolymer gels for nucleotide adsorption. J. Biomater. Sci. Polym. Ed. 2000, 11, 817–831.

    PubMed  Article  CAS  Google Scholar 

  18. [18]

    Ivanov, A. E.; Galaev, I. Y.; Mattiasson, B. Interaction of sugars, polysaccharides and cells with boronate-containing copolymers: From solution to polymer brushes. J. Mol. Recognit. 2006, 19, 322–331.

    PubMed  Article  CAS  Google Scholar 

  19. [19]

    Popii, V.; Baumann, G. Laboratory measurement of growth hormone. Clin. Chim. Acta. 2004, 350, 1–16.

    PubMed  Article  CAS  Google Scholar 

  20. [20]

    Bidlingmaier, M.; Strasburger, C. J. Growth hormone assays: Current methodologies and their limitations. Pituitary 2007, 10, 115–119.

    PubMed  Article  CAS  Google Scholar 

  21. [21]

    Saugy, M.; Robinson, N.; Saudan, C.; Baume, N.; Avois, L.; Mangin, P. Human growth hormone doping in sport. Br. J. Sports Med. 2006, 40Suppl 1, i35–39.

    PubMed  Article  Google Scholar 

  22. [22]

    Ehrnborg, C.; Rosen, T. Physiological and pharmacological basis for the ergogenic effects of growth hormone in elite sports. Asian J. Androl. 2008, 10, 373–383.

    PubMed  Article  CAS  Google Scholar 

  23. [23]

    Barroso, O.; Mazzoni, I.; Rabin, O. Hormone abuse in sports: The antidoping perspective. Asian J. Androl. 2008, 10, 391–402.

    PubMed  Article  CAS  Google Scholar 

  24. [24]

    Rigamonti, A. E.; Cella, S. G.; Marazzi, N.; Di Luigi, L.; Sartorio, A.; Muller, E. E. Growth hormone abuse: Methods of detection. Trends Endocrinol. Metab. 2005, 16, 160–166.

    PubMed  Article  CAS  Google Scholar 

  25. [25]

    Albini, C. H.; Sotos, J.; Sherman, B.; Johanson, A.; Celniker, A.; Hopwood, N.; Quattrin, T.; Mills, B. J.; Macgillivray, M. H. Diagnostic significance of urinary growth hormone measurements in children with growth failure: Correlation between serum and urine growth hormone. Pediatr. Res. 1991, 29, 619–622.

    PubMed  Article  CAS  Google Scholar 

  26. [26]

    Hourd, P.; Edwards, R. Current methods for the measurement of growth hormone in urine. Clin. Endocrinol. (Oxf) 1994, 40, 155–170.

    Article  CAS  Google Scholar 

  27. [27]

    Butt, D. A.; Sochett, E. B. Urinary growth hormone: A screening test for growth hormone sufficiency. Clin. Endocrinol. (Oxf) 1997, 47, 447 454.

    Article  Google Scholar 

  28. [28]

    Saugy, M.; Cardis, C.; Schweizer, C.; Veuthey, J. L.; Rivier, L. Detection of human growth hormone doping in urine: Out of competition tests are necessary. J. Chromatogr. B Biomed. Appl. 1996, 687, 201–211.

    PubMed  Article  CAS  Google Scholar 

  29. [29]

    Denizli, A.; Piskin, E. Dye-ligand affinity systems. J. Biochem. Bioph. Meth. 2001, 49, 391–416.

    Article  CAS  Google Scholar 

  30. [30]

    Wakui, H.; Kobayashi, R.; Itoh, H.; Imai, H.; Nakamoto, Y.; Miura, A. B. High-yield purification of the complex-forming glycoprotein in urine from normal and abnormal subjects. Clin. Chem. 1989, 35, 577–581.

    PubMed  CAS  Google Scholar 

  31. [31]

    Sereikaite, J.; Bumelis, V. A. Examination of dye-protein interaction by gel-permeation chromatography. Biomed. Chromatogr. 2006, 20, 195–199.

    PubMed  Article  CAS  Google Scholar 

  32. [32]

    Sutkeviciute, I.; Sereikaite, J.; Bumelis, V. A. Analysis of cibacron Blue F3G-A interaction with therapeutic proteins by MALDI-TOF mass spectrometry. Biomed. Chromatogr. 2008, 22, 1001–1007.

    PubMed  Article  CAS  Google Scholar 

  33. [33]

    Lee, H. L.; Eom, H. S.; Yun, T.; Kim, H. J.; Park, W. S.; Nam, B. H.; Moon-Woo, S.; Lee, D. H.; Kong, S. Y. Serum and urine levels of interleukin-8 in patients with non-Hodgkin’s lymphoma. Cytokine 2008, 43, 71–75.

    PubMed  Article  CAS  Google Scholar 

  34. [34]

    Huang, G.; Gao, J.; Hu, Z.; St John, J. V.; Ponder, B. C.; Moro, D. Controlled drug release from hydrogel nanoparticle networks. J. Control. Release 2004, 94, 303–311.

    PubMed  Article  CAS  Google Scholar 

  35. [35]

    Pecora, R. Dynamic light scattering: Applications of photo correlation spectroscopy; Springer: 1985.

  36. [36]

    Jin, S. H.; Lee, Y. Y.; Kang, H. Y. Methyl-betacyclodextrin, a specific cholesterol-binding agent, inhibits melanogenesis in human melanocytes through activation of ERK. Arch. Dermatol. Res. 2008, 300, 451–454.

    PubMed  Article  CAS  Google Scholar 

  37. [37]

    Cai, W.; Yao, X.; Shao, X.; Pan, Z. Bimodal complexations of steroids with cyclodextrins by a flexible docking algorithm. J. Incl. Phenom. Macrocycl. Chem. 2005, 51, 41–51.

    Article  CAS  Google Scholar 

  38. [38]

    Borst, C.; Holzgrabe, U. Enantioseparation of dopa and related compounds by cyclodextrin-modified microemulsion electrokinetic chromatography. J. Chromatogr. A 2008, 1204, 191–196.

    PubMed  Article  CAS  Google Scholar 

  39. [39]

    Ugurel, S.; Rappl, G.; Tilgen, W.; Reinhold, U. Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J. Clin. Oncol. 2001, 19, 577–583.

    PubMed  CAS  Google Scholar 

  40. [40]

    Xie, K. Interleukin-8 and human cancer biology. Cytokine Growth Factor Rev. 2001, 12, 375–391.

    PubMed  Article  CAS  Google Scholar 

  41. [41]

    Ren, Y.; Poon, R. T.; Tsui, H. T.; Chen, W. H.; Li, Z.; Lau, C.; Yu, W. C.; Fan, S. T. Interleukin-8 serum levels in patients with hepatocellular carcinoma: Correlations with clinicopathological features and prognosis. Clin. Cancer Res. 2003, 9, 5996–6001.

    PubMed  CAS  Google Scholar 

  42. [42]

    Gall, M. A.; Hougaard, P.; Borch-Johnsen, K.; Parving, H. H. Risk factors for development of incipient and overt diabetic nephropathy in patients with non-insulin dependent diabetes mellitus: Prospective, observational study. BMJ 1997, 314, 783–788.

    PubMed  CAS  Google Scholar 

  43. [43]

    Carroll, M. F.; Temte, J. L. Proteinuria in adults: A diagnostic approach. Am. Fam. Physician 2000, 62, 1333–1340.

    PubMed  CAS  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Alessandra Luchini.

Rights and permissions

This article is published under an open access license. Please check the 'Copyright Information' section either on this page or in the PDF for details of this license and what re-use is permitted. If your intended use exceeds what is permitted by the license or if you are unable to locate the licence and re-use information, please contact the Rights and Permissions team.

About this article

Cite this article

Fredolini, C., Meani, F., Alex Reeder, K. et al. Concentration and preservation of very low abundance biomarkers in urine, such as human growth hormone (hGH), by Cibacron Blue F3G-A loaded hydrogel particles. Nano Res. 1, 502–518 (2008).

Download citation


  • Urine proteomics
  • Cibacron Blue F3G-A
  • human growth hormone
  • hydrogel nanoparticles
  • N-isopropylacrylamide