A simple fluorescence quenching method for the quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacterial infections, is described, where material and reagent costs are minimal. The study harnesses a fluorescence quenching between a nonfluorescent polyelectrolyte containing a ligand (O-phosphorylethanolamine, PEA) and fluorophore (fluorescamine isomer 1) containing polyelectrolyte. The quenching was attributed due to strong polymer-polymer interaction through intermolecular hydrogen bonding. The nonlinear behaviour of Stern-Volmer plot indicates a binding induced quenching, that is, static quenching. However, fluorescence was found to increase in presence of C-reactive protein, due to the specific molecular recognition occurring between CRP and PEA, thereby excluding fluorophore containing chain. A definite correlation was found between concentration of CRP and fluorescence intensity and the method exhibited a linear relationship in the range of 40–360？ng/mL with a detection limit of 30 ± 2？ng/mL. The antibody free method was successfully applied for the analysis of CRP in human serum samples and the method showed good correlation with hospital measurements . Thus the fluorescence based polyelectrolyte biosensor is a potential system for rapid, and antibody free platform for CRP detection. 1. Introduction Polyelectrolytes based fluorescent sensors have been an area of intense research for the past few decades, due to the interest in area such as controlled drug release, surface coating, and chemical and biological sensing [1–5]. Conjugated polyelectrolytes have received particular attention for sensing proteins, due to their water solubility and fluorescence properties [6–9]. Conjugated polyelectrolytes contain a set of structural attributes that make them useful in optical and electronic detection of many chemical and biological targets [10]. Fluorescence methods are being used for a variety of investigations in biochemical, medical, and chemical research. Fluorescence based sensors may rely on changes in emission intensity, life time, and wavelength (excitation and emission). However, fluorescence quenching, in which decrease of emission intensity and/or life time is measured, is one of the most common techniques in fluorescence based sensors because of its easy measurement and low detection limit. Herein we report a nonconjugated polyelectrolyte as efficient fluorescent quencher and its application for the quantitation in serum of an acute phase reactant, C-reactive protein (CRP).
References
[1]
M. Schlupp, T. Weil, A. J. Berresheim, U. M. Wiesler, J. Bargon, and K. Muller, “Polyphenylene dendrimers as sensitive and selective sensor layers,” Angewandte Chemie International Edition, vol. 40, pp. 4011–4015, 2001.
[2]
D. Tyler McQuade, A. E. Pullen, and T. M. Swager, “Conjugated polymer-based chemical sensors,” Chemical Reviews, vol. 100, no. 7, pp. 2537–2574, 2000.
[3]
L. A. Samuelson, D. L. Kaplan, J. O. Lim, M. Kamath, K. A. Marx, and S. K. Tripathy, “Molecular recognition between a biotinylated polythiophene copolymer and phycoerythrin utilizing the biotin-streptavidin interaction,” Thin Solid Films, vol. 242, no. 1-2, pp. 50–55, 1994.
[4]
K. Fa？d and M. Leclerc, “Functionalized regioregular polythiophenes: towards the development of biochromic sensors,” Chemical Communications, no. 24, pp. 2761–2762, 1996.
[5]
M. Hiller, C. Kranz, J. Huber, P. Bauerle, and W. Schuhmann, “Amperometric biosensors produced by immobilization of redox enzymes at polythiophene-modified electrode surfaces,” Advanced Materials, vol. 8, pp. 219–222, 1996.
[6]
G. Sukhorukov, A. Fery, and H. M？hwald, “Intelligent micro- and nanocapsules,” Progress in Polymer Science, vol. 30, no. 8-9, pp. 885–897, 2005.
[7]
C. S. Peyratout and L. Daehne, “Tailor-made polyelectrolyte microcapsules: from multilayers to smart containers,” Angewandte Chemie International Edition, vol. 43, pp. 3672–3678, 2004.
[8]
C. K. Ober and G. Wegner, “Polyelectrolyte-surfactant complexes in the solid state: facile building blocks for self-organizing materials,” Advanced Materials, vol. 9, no. 1, pp. 17–31, 1997.
[9]
P. T. Hammond, “Form and function in multilayer assembly: new applications at the nanoscale,” Advanced Materials, vol. 16, no. 15, pp. 1271–1293, 2004.
[10]
Q. Zhou and T. M. Swager, “Fluorescent chemosensors based on energy migration in conjugated polymers: the molecular wire approach to increased sensitivity,” Journal of the American Chemical Society, vol. 117, no. 50, pp. 12593–12602, 1995.
[11]
J. E. Volanaskis, “Human C-reactive protein: expression, structure, and function,” Molecular Immunology, vol. 38, pp. 189–197, 2001.
[12]
I. Kushner, M. L. Broder, and D. Karp, “Control of the acute phase response. Serum C-reactive protein kinetics after acute myocardial infarction,” Journal of Clinical Investigation, vol. 61, no. 2, pp. 235–242, 1978.
[13]
Q. Zen, W. Zhong, and R. F. Mortensen, “Binding site on human C-reactive Protein (CRP) recognized by the Leukocyte CRP-receptor,” Journal of Cellular Biochemistry, vol. 64, pp. 140–151, 1997.
[14]
M. O. Pentik？inen, K. ？？rni, M. Ala-Korpela, and P. T. Kovanen, “Modified LDL—trigger of atherosclerosis and inflammation in the arterial intima,” Journal of Internal Medicine, vol. 247, no. 3, pp. 359–370, 2000.
[15]
T. B. Ledue, S. E. Poulin, L. F. Leavitt, and A. M. Johnson, “Evaluation of a particle-enhanced immunoassay for quantifying C-reactive protein,” Clinical Chemistry, vol. 35, no. 9, pp. 2001–2002, 1989.
[16]
S. Birnbaum, C. Uden, C. G. M. Magnusson, and S. Nilsson, “Latex-based thin-layer immunoaffinity chromatography for quantitation of protein analytes,” Analytical Biochemistry, vol. 206, no. 1, pp. 168–171, 1992.
[17]
S. Nilsson, C. Lager, T. Laurell, and S. Birnbaum, “Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein,” Analytical Chemistry, vol. 67, no. 17, pp. 3051–3056, 1995.
[18]
X. Chu, J. Jiang, G. Shen, and R. Yu, “Simultaneous immunoassay using piezoelectric immunosensor array and robust method,” Analytica Chimica Acta, vol. 336, no. 1–3, pp. 185–193, 1996.
[19]
R. E. Banks, “Measurement of cytokines in clinical samples using immunoassays: problems and pitfalls,” Critical Reviews in Clinical Laboratory Sciences, vol. 37, no. 2, pp. 131–182, 2000.
[20]
J. L. Bock, “The new era of automated immunoassay,” American Journal of Clinical Pathology, vol. 113, no. 5, pp. 628–646, 2000.
[21]
P. M. Ridker, M. Cushman, M. J. Stampfer, R. P. Tracy, and C. H. Hennekens, “Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease,” Circulation, vol. 97, no. 5, pp. 425–428, 1998.
[22]
C. M. Merritt and J. W. Winkelman, “Electrochemical method for measuring C-reactive protein using crown ether-phosphate ester ionophores,” Analytical Chemistry, vol. 61, no. 21, pp. 2362–2365, 1989.
[23]
M. Sivakumar and K. Panduranga Rao, “In vitro release of ibuprofen and gentamicin from PMMA functional microspheres,” Journal of Biomaterials Science, Polymer Edition, vol. 13, no. 2, pp. 111–126, 2002.
[24]
Y. Takeoka, K. Sasada, Y. Nishiwaki, M. Rikukawa, and K. Sanui, “Fabrication of polycondensed multilayer thin films by a self-assembly method,” Colloids and Surfaces A, vol. 257-258, pp. 485–488, 2005.
[25]
J. B. Lee, D.-J. Kim, J.-W. Choi, and K.-K. Koo, “Formation of a protein monomolecular layer by a combined technique of LB and SA methods,” Colloids and Surfaces B, vol. 41, no. 2-3, pp. 163–168, 2005.
[26]
V. Kabanov, A. Zezin, V. Izumrodov, T. Bronich, and K. Bakeev, “Cooperative interpolyelectrolyte reactions,” Die Makromolekulare Chemie, vol. 13, pp. 137–155, 1985.
[27]
S. J. Dwight, B. S. Gaylord, J. W. Hong, and G. C. Bazan, “Perturbation of fluorescence by nonspecific interactions between anionic poly(phenylenevinylene)s and proteins: implications for biosensors,” Journal of the American Chemical Society, vol. 126, no. 51, pp. 16850–16859, 2004.
[28]
S. Shi and F. Wudl, “Synthesis and characterization of a water-soluble poly(p-phenylenevinylene) derivative,” Macromolecules, vol. 23, no. 8, pp. 2119–2124, 1990.
[29]
J. Park, S. Kurosawa, J. Watanabe, and K. Ishihara, “Evaluation of 2-methacryloyloxyethyl phosphorylcholine polymeric nanoparticle for immunoassay of C-reactive protein detection,” Analytical Chemistry, vol. 76, no. 9, pp. 2649–2655, 2004.
