Nano Research

, Volume 1, Issue 6, pp 502–518 | Cite as

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

  • Claudia Fredolini
  • Francesco Meani
  • K. Alex Reeder
  • Sally Rucker
  • Alexis Patanarut
  • Palma J. Botterell
  • Barney Bishop
  • Caterina Longo
  • Virginia Espina
  • Emanuel F. PetricoinIII
  • Lance A. Liotta
  • Alessandra LuchiniEmail author
Open Access
Research Article


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.


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


  1. [1]
    Gonzalez-Buitrago, J. M.; Ferreira, L.; Lorenzo, I. Urinary proteomics. Clin. Chim. Acta 2007, 375, 49–56.PubMedCrossRefGoogle 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.PubMedGoogle Scholar
  3. [3]
    Pisitkun, T.; Johnstone, R.; Knepper, M. A. Discovery of urinary biomarkers. Mol. Cell. Proteomics 2006, 5, 1760–1771.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  5. [5]
    Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Nanomedicine: Current status and future prospects. Faseb. J. 2005, 19, 311–330.PubMedCrossRefGoogle Scholar
  6. [6]
    Jain, K. K. Applications of nanobiotechnology in clinical diagnostics. Clin. Chem. 2007, 53, 2002–2009.PubMedCrossRefGoogle 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.PubMedCrossRefADSGoogle Scholar
  8. [8]
    Jones, C. D.; Lyon, L. A. Synthesis and characterization of multiresponsive core-shell microgels. Macromolecules 2000, 33, 8301–8306.CrossRefGoogle 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.CrossRefGoogle Scholar
  10. [10]
    Suzuki, A. and Tanaka, T. Phase transition in polymer gels induced by visible light. Nature 1990, 346, 345–347.CrossRefADSGoogle 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.PubMedCrossRefADSGoogle Scholar
  12. [12]
    Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 2002, 54, 3–12.PubMedCrossRefGoogle 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.CrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  17. [17]
    Tuncel, A.; Ozdemir, A. Thermally reversible VPBANIPAM copolymer gels for nucleotide adsorption. J. Biomater. Sci. Polym. Ed. 2000, 11, 817–831.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  19. [19]
    Popii, V.; Baumann, G. Laboratory measurement of growth hormone. Clin. Chim. Acta. 2004, 350, 1–16.PubMedCrossRefGoogle Scholar
  20. [20]
    Bidlingmaier, M.; Strasburger, C. J. Growth hormone assays: Current methodologies and their limitations. Pituitary 2007, 10, 115–119.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  23. [23]
    Barroso, O.; Mazzoni, I.; Rabin, O. Hormone abuse in sports: The antidoping perspective. Asian J. Androl. 2008, 10, 391–402.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  26. [26]
    Hourd, P.; Edwards, R. Current methods for the measurement of growth hormone in urine. Clin. Endocrinol. (Oxf) 1994, 40, 155–170.CrossRefGoogle 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.CrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  29. [29]
    Denizli, A.; Piskin, E. Dye-ligand affinity systems. J. Biochem. Bioph. Meth. 2001, 49, 391–416.CrossRefGoogle 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.PubMedGoogle Scholar
  31. [31]
    Sereikaite, J.; Bumelis, V. A. Examination of dye-protein interaction by gel-permeation chromatography. Biomed. Chromatogr. 2006, 20, 195–199.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedCrossRefGoogle Scholar
  35. [35]
    Pecora, R. Dynamic light scattering: Applications of photo correlation spectroscopy; Springer: 1985.Google Scholar
  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.PubMedCrossRefGoogle 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.CrossRefGoogle 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.PubMedCrossRefGoogle 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.PubMedGoogle Scholar
  40. [40]
    Xie, K. Interleukin-8 and human cancer biology. Cytokine Growth Factor Rev. 2001, 12, 375–391.PubMedCrossRefGoogle 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.PubMedGoogle 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.PubMedGoogle Scholar
  43. [43]
    Carroll, M. F.; Temte, J. L. Proteinuria in adults: A diagnostic approach. Am. Fam. Physician 2000, 62, 1333–1340.PubMedGoogle Scholar

Copyright information

© Tsinghua Press and Springer-Verlag GmbH 2008

Authors and Affiliations

  • Claudia Fredolini
    • 1
    • 2
    • 5
  • Francesco Meani
    • 3
    • 5
  • K. Alex Reeder
    • 5
  • Sally Rucker
    • 5
  • Alexis Patanarut
    • 6
  • Palma J. Botterell
    • 6
  • Barney Bishop
    • 6
  • Caterina Longo
    • 4
    • 5
  • Virginia Espina
    • 5
  • Emanuel F. PetricoinIII
    • 5
  • Lance A. Liotta
    • 5
  • Alessandra Luchini
    • 5
    Email author
  1. 1.Department of UrologyS. Giovanni Bosco HospitalTorinoItaly
  2. 2.Department of Medicine and Experimental OncologyUniversity of TurinTurinItaly
  3. 3.Gynecology and Obstetrics DepartmentUniversity of BresciaBresciaItaly
  4. 4.Department of Dermatology and VenereologyUniversity of Modena and Reggio EmiliaModenaItaly
  5. 5.Center for the Applied Proteomics and Molecular MedicineGeorge Mason UniversityManassasUSA
  6. 6.Chemistry and Biochemistry DepartmentGeorge Mason UniversityManassasUSA

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