Langmuir 1995,11,1261-1267
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Adsorption and Desorption Studies of Polyadenylic Acid onto Positively Charged Latex Particles Abdelhamid Elaissari,t Christian Pichot,*$?Thierry Delair,? Philippe Cros,* and Robin Kiirfurstt Unit4 mixte CNRS-BioMirieux, Laboratoire des sondes IzuclCiques, BioMkrieux, Ecole Normale Supdrieure de Lyon, 46,Allke d'ltalie 69364 Lyon Cedex 07,France Received April 19, 1994. I n Final Form: December 29, 1994@ The adsorption of single-stranded polyadenylic acid (poly dA)(30 bases) onto cationic polystyrene latex particles was studied. The latex particles were prepared by soap-free emulsion copolymerization of styrene and vinyl benzylamine hydrochloride. Adsorption experiments were carried out using a fluoresceinlabeled polyadenylic acid and adsorption isotherms were established for constant ionic strength in a pH range of 4-9. Poly dA adsorption was optimal at low pH and decreased upon increasing pH. Desorption of oligodeoxyribonucleotides was studied by washing the latex particles and by changing the pH. The adsorption of polyadenylic acid onto cationic latexes is interpreted based upon the contributions of hydrophobic and electrostatic interactions.
Introduction Nucleic acid hybridization techniques are widely used in the detection of specific complementary nucleic acid sequences in molecular biology and applied fields, such as diagnostic medicine. Nitrocellulose or nylon membrane filters are typically used as solid supports in hybridization. However this method is limited by the low rate of nucleic acids in solution hybridizing to the nucleic acids covalently bound to these solid support^.^ Microspheric polymeric supports, on the other hand, such as polystyrene latex2 beads, have excellent hybridization kinetics. Moreover, their size and functionality can easily be modified and they offer low cost production. A number of methods have been published for the immobilization of oligodeoxyribonucleotides on such s u p p ~ r t s , lbut ~ ~this ~ ~ methodology -~~ can lead to high levels of nonspecific oligodeoxyribonucleotide adsorption. No systematic study has appeared on the adsorption characteristics of nucleic acids onto latex particles. In one study the adsorption of large deoxyribonucleic acids onto polystyrene latex particledl was described. This study examined the adsorption of native and heatdenatured calf thymus DNA and poly(cytidy1ic acid) poly(inosinic acid) onto negatively charged polystyrene latex particles. Since the use of oligodeoxyribonucleotideprobes has advantages over large nucleic acid fragments, due to their more rapid kinetics of hybridization and since, by using DNA synthesizers, it is possible to obtain large ~~~
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Unit6 mixte CNRS-BioMBrieux. * Laboratoire des sondes nucleiques, BioM6riew. Abstract publishedinAdvance ACSAbstracts, March 15,1995. +
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(1) Wolf, S. T.; Haines, L.; Fisch, J.; Kremsky, J. N.; Dougherty, J. P.; Jacobs, K. Nucleic Acid. Res. 1987,15,2911. (2) Hara, E.; Yamaguchi, T.; Tahara, H.; Tsuyama, N.; Tsurui, H.; Ide, T.; Oda, K. Anal. Biochem. 1993,214,58. (3) Delair, Th.; Marguet, V.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1994,342. (4) Delair, Th.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1993, 271. (5) Van Ness, J.;Kalbfleisch, S.; Petrie, C. R.; Reed, M. W.; Tabone, J. C.; Vermeulen, N. M. J. Nucleic Acid Res. 1991,19,3345. (6) Maskos, U.; Southern, E. M. Nucleic Acid Res. 1992,20,1679. (7) Fahy, E.;Davis, G. R.;Dimichele,L. J.;Ghosh, S. S.NucleicAcid. Res. 1993,21,1818. (8) Day, P. J. R.; Flora, P. S.; Fox, J. E.; Walker, M. R. Biochem. J . 1991,278,735. (9) Albertsen, C.; Kalland, K. H.; Haukanes, B. I.; Havarstein, L. S.; Kleppe, K. Anal. Biochem. 1990,189,40. (10)Ghosh, S. S.; Musso, G. F. Nucleic Acid. Res. 1987,15,5353. (11) Yamaoka, K.; Fukudome, K.; Mukaiyama, N.; Shirahama, H.; Suzawa, T. J . Colloid Interface Sci. 1990,136, 519.
quantities of well-defined, high-purity oligodeoxyribonucleotides after reverse phase chromatography, we focused our attention on the study of the adsorption ofthe short oligodeoxyribonucleotides ( 150 bases) onto latex particles. A preliminary study investigating the adsorption of single-strandedoligodeoxyribonucleotidecontaining three nucleotides, T, G, and C, onto anionic and cationic submicronic size polystyrene particles has appeared.12 However, for a better understanding of this phenomenon, it was of particular interest to consider the adsorption characteristics of homopolynucleic acids. The study is specifically devoted to the adsorptioddesorptionproperties of polyadenylic acid (30 bases) onto positively charged latex particles. The characteristics of the monodispersed amine-charged polystyrene latex particles and the polynucleotide are first reviewed, then we report on the adsorption behavior of the single-stranded polyadenylic acid as a function of the pH of the incubation medium.
Materials and Methods 1. Synthesis of the Polyadenylic Acid Labeled with a Fluorescein Group. The single-stranded oligodeoxyribonucle-
otide d(A)l~,d(U*)d(A)14 was synthesized on an automatic DNA/ RNA synthesizer (model 394 from Applied Biosystems) using 2-cyanoethyl phosphoramidite chemistry. To label the oligodeoxyribonucleotide, the phosphoramidite derivative of uracil substituted by the method of Ruth13 was incorporated at the 16th position. This modified nucleoside exhibits,on the C5 ofthe base, a linker bearing a terminal primary amine function protected by an alkaline labile group. After oligodeoxyribonucleotide synthesis, treatment with ammonia gave several fully deprotected compounds from which the desired product was purified using reverse phase chromatography. Coupling to fluorescein was carried out as follows. The modified oligodeoxyribonucleotide(67 nmol) was dissolved in 1mL of 0.2 M sodium hydrogen carbonate,0.15 M NaCl buffer, pH 8.8,and then added to 200 mL of a 32 mM dimethylformamide solution of fluorescein isothiocyanate (FITC),isomer I (Aldrich). After 2 h at 50 "C excessfluorescein isothiocyanatewas destroyed by adding 25 mL of a 1 M ammonium chloride solution. Fluorescein-conjugatedoligonucleotidewas precipitated with ethyl alcohol and purified by reverse-phase ~hromatography.'~ (12) Eldssari, A.; Cros, P.; Pichot, C.; Laurent, V.; Mandrand, B. Colloids Surf. 1994,83, 25. (13) Ruth, J. L. DNA 1984,3, 123. (14) Caruthers, M. H.; Beaton, G.; Wu, J. U.; Wiesler, W. Methods Enzymol. 1992,211,3.
0743-7463/95/2411-1261$09.00/0 0 1995 American Chemical Society
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The average yield of the labeling reaction was about 50%. The product was quantitated by UV absorption a t 260 nm. Equation 1 was used to determine the concentration:
-c_1
A260
(6AnA + 6U*nU1+ CFnF)
(1)
where C is the concentration of the oligodeoxyribonucleotide in mole dm-3, A260 is the absorption at 260 nm, and TZA,nu, and n~ are the number of nucleosides and fluorophore probes, respectively, present in t h e sequence, and I is the length ofthe cell. The following molar extinction coefficients were used: E A = 14 500, EU* = 9200, and CF = 17 900 mol-' cm-2. The molecular weight (M, = 10 200 g mol-') of the conjugate was calculated by using standard formula.l5 2. Preparation and Characterization of Polymer Latex Particles. Unless otherwise stated, reagents were used as received. Water was Milli-Q grade (Millipore SA) and was boiled for 1 h under a nitrogen stream before use. Styrene (Janssen Chemica) was distilled under reduced pressure, 2,2'-azobis(2amidinopropane) dihydrochloride (V-501, kindly provided by Wako Chemicals Gmbh was recrystallized from water-acetone. Divinylbenzene (DVB)(Aldrich) was used as such. The comonomer (VBAH) was prepared by a standard synthetic procedure.16 Stable cationic latex was prepared by emulsifier-free emulsion copolymerization of styrene and vinylbenzylamine hydrochloride (VBAH) using 2,2'-azobis(2-amidinopropane)hydrochloride (V50) as the initiator. Monodispersity of the particles size was improved by using divinylbenzene (DVB)as a third monomer at a 2% molar ratio. The polymerization was carried out in a 250 mL thermostated glass reactor, equipped with anchor-shaped stirrer, condenser, thermocouple, and a gas inlet and outlet. The polymerization was carried out under nitrogen, using a stirrer adjusted at 300 rpm, and the temperature was kept constant at 80 "C. A typical latex synthesis reaction consisted of H20, 180 g; styrene, 10 g; V-50, 0.14 g; VBAH, 0.158 g; and DVB, 0.219 g. VBAH and 2,2'-azobis(2-amidinopropane)are responsible for the surface charges on the latexparticles. Surface charge density measurement (concentration of NH2 on the particle surface) was determined by a UV spectrometry method with N-succinimidyl 3-(2-pyridyldithio)propionate(SPDP).3J6 Latex particle size and distribution was determined by transmission electron microscopy (TEM) (from Hitachi) a t the C W O , Universite Claude Bernard, Lyon I, France. Particles were dried on a carbon-coated copper grid at room temperature and the size was determined from TEM micrographs using a Hewlett-Packard 911 A digitalizer which gave the number (D,) and weight (D,)average particle diameters. The zeta-potential17J8(5) oflatex particles was measured using a Zeta Sizer I11 (from Malvern Instruments), equipped with a laser as light source.
