Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining them in situ. 1. Introduction Glass is essentially an amorphous 3-dimensional mesh of silica oxides terminating at the surface as silicon hydroxides. Although the surface exposed silica hydroxides readily coordinate with divalent cations such as Ca2+ and can promote surface activated plasma coagulation [1], glass is surprisingly resistant to protein adsorption. For example, glass bead chromatography has been used to specifically purify vitronectin, a cell adhesion protein, from serum with a high degree of purity [2]. Vitronectin has an arginine-glycine-aspartic acid (RGD) integrin recognition motif and was initially identified as “serum spreading factor” due to its ability to bind tissue culture plates and mediate cell adhesion and spreading [3]. Additionally, glass beads can be functionalized with a variety of silanes containing amine and thiol functional groups that can be used for biomolecular conjugation [4, 5]. However, development of porous polymer resins with superior flow characteristics and greater surface area have since replaced glass beads as the solid support of choice for most protein purification. In this study, we revisit glass chromatography by examining the ability of hollow glass microbubbles to isolate proteins, or cells, from serum and blood. Glass bubbles are inexpensive, lightweight, and strong hollow silica spheres that are commonly
References
[1]
J. Margolis, “Activation of plasma by contact with glass: evidence for a common reaction which releases plasma kinin and initiates coagulation,” The Journal of Physiology, vol. 144, no. 1, pp. 1–22, 1958.
[2]
D. W. Barnes and J. Silnutzer, “Isolation of human serum spreading factor,” Journal of Biological Chemistry, vol. 258, no. 20, pp. 12548–12552, 1983.
[3]
E. G. Hayman, M. D. Pierschbacher, Y. Ohgren, and E. Ruoslahti, “Serum spreading factor (vitronectin) is present at the cell surface and in tissues,” Proceedings of the National Academy of Sciences of the United States of America, vol. 80, no. 13, pp. 4003–4007, 1983.
[4]
P. Cuatrecasas, “Affinity chromatography,” Annual Review of Biochemistry, vol. 40, pp. 259–278, 1971.
[5]
S. K. Bhatia, L. C. Shriver-Lake, K. J. Prior et al., “Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces,” Analytical Biochemistry, vol. 178, no. 2, pp. 408–413, 1989.
[6]
J. N. Adkins, S. M. Varnum, K. J. Auberry et al., “Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry,” Molecular & Cellular Proteomics, vol. 1, no. 12, pp. 947–955, 2002.
[7]
N. L. Anderson and N. G. Anderson, “The human plasma proteome: history, character, and diagnostic prospects,” Molecular & Cellular Proteomics, vol. 1, no. 11, pp. 845–867, 2002.
[8]
N. Tang, P. Tornatore, and S. R. Weinberger, “Current developments in SELDI affinity technology,” Mass Spectrometry Reviews, vol. 23, no. 1, pp. 34–44, 2004.
[9]
Q. Liu, F. Xie, A. Alvarado-Diaz et al., “Neurofilamentopathy in neurodegenerative diseases,” Open Neurology Journal, vol. 5, no. 1, pp. 58–62, 2011.
[10]
D. M. Toivola, P. Strnad, A. Habtezion, and M. B. Omary, “Intermediate filaments take the heat as stress proteins,” Trends in Cell Biology, vol. 20, no. 2, pp. 79–91, 2010.
[11]
N. Norgren, L. Rosengren, and T. Stigbrand, “Elevated neurofilament levels in neurological diseases,” Brain Research, vol. 987, no. 1, pp. 25–31, 2003.
[12]
J. Brettschneider, A. Petzold, S. D. Sü？muth et al., “Neurofilament heavy-chain NfHSMI35 in cerebrospinal fluid supports the differential diagnosis of parkinsonian syndromes,” Movement Disorders, vol. 21, no. 12, pp. 2224–2227, 2006.
[13]
J. Brettschneider, A. Petzold, D. Sch？ttle, A. Claus, M. Riepe, and H. Tumani, “The neurofilament heavy chain ( ) in the cerebrospinal fluid diagnosis of Alzheimer's disease,” Dementia and Geriatric Cognitive Disorders, vol. 21, no. 5-6, pp. 291–295, 2006.
[14]
A. Antinori, G. Arendt, J. T. Becker, B. J. Brew, D. A. Byrd, and D. B. Clifford, “Biomarkers of HIV-associated neurocognitive disorders,” in Proceedings of the HIV Infection and the Central Nervous System: Developed and Resource Limited Settings, Rome, Italy, June 2005.
[15]
M. Gisslén, L. Rosengren, L. Hagberg, S. G. Deeks, and R. W. Price, “Cerebrospinal fluid signs of neuronal damage after antiretroviral treatment interruption in HIV-1 infection,” AIDS Research and Therapy, vol. 2, no. 1, article 6, 2005.
[16]
P. Zhu, D. R. Shelton, S. Li et al., “Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay,” Biosensors and Bioelectronics, vol. 30, no. 1, pp. 337–341, 2011.
[17]
S. Li, Y. Zhang, P. Amstutz III, and C.-M. Tang, “Multiplex integrating waveguide sensor: signalyte-II,” Methods in Molecular Biology, vol. 503, pp. 423–434, 2009.
[18]
S. Li, L. Kiefer, P. Zhu et al., “Ultra-sensitive detection using integrated waveguide technologies,” in Biosensors For Health, Environment and Biosecurity/Book 3, Intech, Rijeka, Croatia, 2011.
[19]
H. M. Poehling and V. Neuhoff, “Visualization of proteins with a silver “stain”: a critical analysis,” Electrophoresis, vol. 2, pp. 141–147, 1981.
[20]
L. Trinkle-Mulcahy, S. Boulon, Y. W. Lam et al., “Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes,” Journal of Cell Biology, vol. 183, no. 2, pp. 223–239, 2008.
[21]
S. Stefansson, D. Adams, and C. M. Tang, “Sample preparation using buoyant silica microspheres,” in Proceedings of the 6th Early Detection Research Network Workshop (EDRN '09), Bethesda, Md, USA, August-September 2009.
[22]
C. H. Hsu, C. C. Chen, D. Irimia, and M. Toner, “Isolating cells from blood using Buoyancy Activated Cell Sorting (BACS) with glass microbubbles,” in Proceedings of the 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Groningen, The Netherlands, October 2010.
