Langmuir 1995,11, 2423-2428
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Fluorescence Energy Transfer from Fluorescein to TetramethylrhodamineCovalently Bound to the Surface of Polystyrene Latex Particles Marie-Th6rBse Charreyre, Ahmad Yekta, and Mitchell A. Winnik" Department of Chemistry and Erindale College, University of Toronto, 80 St. George Street, Toronto, Ontario M5S lAl, Canada
Thierry Delair and Christian Pichot Unite Mixte CNRS-BioMirieux, ENSL, 46 A116e d'ltalie, 69364 Lyon cedex 07, France Received October 20, 1994. I n Final Form: April 5, 1995@ Steady-state fluorescence energy transfer experiments have been carried out with fluorescein (donor) and tetramethylrhodamine (acceptor)covalentlybound onto the surface of amino functionalized polystyrene ester were used latex microspheres. 5-Carboxyfluorescein-NHSand 6-carboxytetramethylrhodamine-NHS to synthesize donor-labeled particles and acceptor-labeledparticles respectively, as well as donor-acceptorlabeled particles. Some changes in the spectroscopiccharacteristics of the dyes after their covalent binding onto the PS latex were observed. A theoretical model describing a random distribution of dyes onto the surface of a sphere corroborates well the experimental energy transfer results.
Introduction Fluorescence energy transfer experiments with the dye couple fluorescein- tetramethylrhodamine have been applied extensively in biology to examine the structure of oligonucleotides,'r2 to follow their h y b r i d i ~ a t i o n and , ~ , ~to study aggregation of peptides on a membrane.5,6 The direct nonradiative energy transfer [DET] process is appropriate for studying these systems since it provides information about the distribution or progmity of species over distances in the range of 15-100 A. Latex particles also represent a kind of system in which information on these length scales is needed and can be studied by DET. For example, one is interested in the smoothness or roughness of the particle surface and the distribution of surface functionality. The surface morphology of polystyrene and poly(buty1methacrylate) latex particles has recently been described with success by DET using the dye couples rhodamine Blmalachite green and rhodamine GG/malachite green.7r8 In these experiments, the dyes were simply adsorbed to the particle surface, with adsorption taking place through a combination of Coulombic and hydrophobic interactions between the dyes and the surface. The fluoresceidtetramethylrhodamine [F/TMR] pair has the advantage that dye derivatives are available for covalent attachment of the dyes to functional groups on the latex surface. As we shall see below, this dye pair poses certain problems in trying to study the system by fluorescence decay analysis. Consequently, in this article we present a steady-state analysis of fluorescence energy Abstract published in Advance A C S Abstracts, June 15,1995. (1)Murchie, A. J. H.; Clegg, R. M.; von Kitzing, E.; Duckett, D. R.; Diekmann, S.; Lilley D. M. J. Lett. Nature 1989,341,763. (2) Eis, P. S.; Millar, D. P. Biochemistry 1993,32,13852. (3) Cardullo, R. A,; Agrawal, S.; Flores, C.; Zamecnik, P. C.; Wolf D. E. Proc. Natl. Acad. Sci. 1988,85, 8790. (4)Morrison, L. E.; Stols, L. M.; Biochemistry 1993,32,3095. (5)Rapaport, D.; Shai, Y. J. B i d . Chem. 1992,267,No. 10,6502. (6) Harris, R. W.; Sims, P. J.;Tweten, R. K. J.B i d . Chem. 1991,266, No. 11, 6936. (7) Nakashima, K.; Duhamel, J.;Winnik, M. A. J.Phys. Chem. 1993, @
97, 10702. ( 8 ) Nakashima, K.;Liu, Y. S.; Zhang, Winnik, M. A.Langmuir 1993,9,2825.
P.; Duhamel, J.; Feng, J.;
transfer from F to TMR, with both dyes covalently attached (via their N-hydroxysuccinimide [NHS] derivative) to the surface of a n amino functionalized polystyrene latex.gWe are not aware of any previous experiments to study energy transfer in two dimensions by steady-state methods, and we derive the appropriate equations to describe the efficiency of DET as a function of acceptor concentration.
Experimental Section Reagents. 5-Carboxyfluorescein-NHSester and 6-carboxytetramethylrhodamine-NHS ester were graciously given by Applied Biosystems Inc. and were used as received. The synthesis and characterization of polystyrene latex particles NC9-4 have been described in ref 9. They were narrowly distributed in size, with a number average diameter D, = 454 nm and a polydispersity index of 1.004 (determined by TEM). The surface charge density in amino groups, obtained by conductometrictitration, is of 26 pequivlg dry latex (or 19.5 pCl cm2). Labelingof the Particles. The binding reaction was carried out in a sodium bicarbonate buffer pH 8.2, 100 mM. The latex (0.5 mL, 5 wt % solids) and the buffer (0.50- x mL) were mixed, then an excess of dye dissolved in DMSO (xpL, x = 5-80 pL), was added. Thus the total volume of each sample was 1.0 mL. Each sample was strongly shaken for several minutes. Some reference samples were prepared in parallel with 0.5 mL of deionized water instead of latex. After gentle shaking for 12 h at room temperature, the samples were centrifuged during 12 min at 8000 rpm. The supernatant was filtered (0.22 pm Gelman Acrodisc) and analyzed by fluorescence to determine the concentration of unreacted dye (tetramethylrhodamine I(ex) = 549 nm, ,Uem)= 574 nm; fluorescein I(ex) = 493 nm and l(em) = 518 nm; slit width = 0.5 mm). The emission intensity of each supernatant was compared with that of the corresponding reference sample (whichhad also been filtered);the difference between the amount of dye in the supernatant and the initially introduced amount of dye corresponds to the amount of dye on the surface of the particles. The particles were redispersed in bicarbonate buffer and centrifugedagain to study the phenomenonof dye desorption. Severalcentrifugatiodredispersion cycles were carried out until less than 1%of the initially introduced amount of dye was found in the supernatant. (9) Delair, T.; Marguet, V.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1994,272,962.
0743-746319512411-2423$09.00/0 0 1995 American Chemical Society
2424 Langmuir, Vol. 11, No. 7, 1995
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