Abstract
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.

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Gonzalez-Buitrago, J. M.; Ferreira, L.; Lorenzo, I. Urinary proteomics. Clin. Chim. Acta 2007, 375, 49–56.
Barratt, J.; Topham, P. Urine proteomics: The present and future of measuring urinary protein components in disease. CMAJ 2007, 177, 361–368.
Pisitkun, T.; Johnstone, R.; Knepper, M. A. Discovery of urinary biomarkers. Mol. Cell. Proteomics 2006, 5, 1760–1771.
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.
Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Nanomedicine: Current status and future prospects. Faseb. J. 2005, 19, 311–330.
Jain, K. K. Applications of nanobiotechnology in clinical diagnostics. Clin. Chem. 2007, 53, 2002–2009.
Tanaka, T.; Sato, E.; Hirokawa, Y.; Hirotsu, S.; Peetermans, J. Critical kinetics of volume phase transition of gels. Phys. Rev. Lett. 1985, 55, 2455.
Jones, C. D.; Lyon, L. A. Synthesis and characterization of multiresponsive core-shell microgels. Macromolecules 2000, 33, 8301–8306.
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.
Suzuki, A. and Tanaka, T. Phase transition in polymer gels induced by visible light. Nature 1990, 346, 345–347.
Tanaka, T.; Nishio, I.; Sun, S. -T.; Ueno-Nishio, S. Collapse of gels in an electric field. Science.1982, 218, 467–469.
Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 2002, 54, 3–12.
Liu, J.; Pelton, R.; Hrymak, A. N. Properties of poly (n-isopropylacrylamide)-grafted colloidal silica. J. Colloid Interf. Sci. 2000, 227, 408–411.
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.
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.
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.
Tuncel, A.; Ozdemir, A. Thermally reversible VPBANIPAM copolymer gels for nucleotide adsorption. J. Biomater. Sci. Polym. Ed. 2000, 11, 817–831.
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.
Popii, V.; Baumann, G. Laboratory measurement of growth hormone. Clin. Chim. Acta. 2004, 350, 1–16.
Bidlingmaier, M.; Strasburger, C. J. Growth hormone assays: Current methodologies and their limitations. Pituitary 2007, 10, 115–119.
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.
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.
Barroso, O.; Mazzoni, I.; Rabin, O. Hormone abuse in sports: The antidoping perspective. Asian J. Androl. 2008, 10, 391–402.
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.
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.
Hourd, P.; Edwards, R. Current methods for the measurement of growth hormone in urine. Clin. Endocrinol. (Oxf) 1994, 40, 155–170.
Butt, D. A.; Sochett, E. B. Urinary growth hormone: A screening test for growth hormone sufficiency. Clin. Endocrinol. (Oxf) 1997, 47, 447 454.
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.
Denizli, A.; Piskin, E. Dye-ligand affinity systems. J. Biochem. Bioph. Meth. 2001, 49, 391–416.
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.
Sereikaite, J.; Bumelis, V. A. Examination of dye-protein interaction by gel-permeation chromatography. Biomed. Chromatogr. 2006, 20, 195–199.
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.
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.
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.
Pecora, R. Dynamic light scattering: Applications of photo correlation spectroscopy; Springer: 1985.
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.
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.
Borst, C.; Holzgrabe, U. Enantioseparation of dopa and related compounds by cyclodextrin-modified microemulsion electrokinetic chromatography. J. Chromatogr. A 2008, 1204, 191–196.
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.
Xie, K. Interleukin-8 and human cancer biology. Cytokine Growth Factor Rev. 2001, 12, 375–391.
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.
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.
Carroll, M. F.; Temte, J. L. Proteinuria in adults: A diagnostic approach. Am. Fam. Physician 2000, 62, 1333–1340.
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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). https://doi.org/10.1007/s12274-008-8054-z
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DOI: https://doi.org/10.1007/s12274-008-8054-z