Effect of Triton X-405 on the Adsorption and Desorption of Single

Dec 1, 1997 - cationic particles, showing a maximum amount of ∼0.5 mg m-2 at the plateau. Desorption of the nonionic surfactant even after an extens...
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Langmuir 1997, 13, 7021-7029

7021

Effect of Triton X-405 on the Adsorption and Desorption of Single-Stranded DNA Fragments onto Positively Charged Latex Particles Franc¸ ois Ganachaud, Abdelhamid Elaı¨ssari,* and Christian Pichot Unite´ Mixte CNRS-BioMe´ rieux, Ecole Normale Superieure de Lyon, 46 Alle´ e d’Italie, 69364 Lyon Cedex 07, France Received May 9, 1997. In Final Form: October 20, 1997X The influence of a nonionic surfactant (Triton X-405) on the adsorption behavior of single-stranded oligodeoxyribonucleotide (poly(thymidylic acid) (dT35)) onto cationic aminated polystyrene latex particles has been investigated. A preliminary study was performed on the adsorption of Triton X-405 onto these cationic particles, showing a maximum amount of ∼0.5 mg m-2 at the plateau. Desorption of the nonionic surfactant even after an extensive cleaning procedure was not completed, indicating that probably low HLB (hydrophilic and lipophilic balance) species were still strongly adsorbed. The modification of the electrophoretic mobility of the latex particles together with the slight decrease in the colloidal stability suggested that the residual Triton X-405 molecules would adopt a flat conformation at the particles surface. Then, the adsorption behavior of dT35 onto precoated cationic latex was thoroughly examined under various conditions and compared with the case of bare latex particles. It was clearly evidenced that the residual adsorbed amount of nonionic surfactant significantly affected the surface nature, especially at basic pH. It was interesting to notice that dT35 adsorption was strongly reduced at basic pH and that its desorption was favored using basic pH buffer containing Triton X-405 and high ionic strength. Kinetic exchange experiments were also performed using labeled 32P-dT35 and unlabeled dT35, indicating that the exchange took place with no marked differences between coated and bare latex particles.

I. Introduction Synthetic single-stranded oligonucleotides (ODNs) fixed onto solid-phase supports are now widely used in assays based on the specific detection of complementary target DNA or RNA sequences contained in biological fluids.1-4 Immobilization of nucleic acids onto solid supports can be performed through various methods such as, physical adsorption,5 covalent binding,1 or molecular recognition (biotin-streptavidin6,7 system for instance); however, for preserving the hybridization properties of the probes, special attention should be paid to control the location and the conformation of the oligonucleotides at the interface after the immobilization step. Due to their polyelectrolyte properties, nucleic acids can interact with supports through different forces (electrostatic, hydrophobic, hydrogen bonding, etc.). In any case, the ODN adsorption usually competes with other immobilization processes; therefore, it is of paramount importance to get qualitative and quantitative information on their adsorption as a function of the type of support (especially the nature of the interface) and experimental conditions (pH, ionic strength, buffer, presence of surfactant, etc.). In our previous studies that dealt with the adsorption of various oligonucleotides onto positively charged poly* To whom correspondence should be addressed. E-Mail: [email protected]. X Abstract published in Advance ACS Abstracts, December 1, 1997. (1) Imai, T.; Sumi, Y.; Hatakeyama, M.; Fujimoto, K.; Kawaguchi, H.; Hayashida, N.; Shiozaki, K.; Terada, K.; Yajima, H.; Handa, H. J. Colloid Interface Sci. 1996, 177, 245. (2) Inomata, Y.; Wada, T.; Handa, H.; Fujimoto, K. Kawaguchi, H. J. Biomater. Sci., Polym. Ed. 1994, 5, 293. (3) Walker, G. T.; Linn, C. P.; Nadeau, Nucleic Acids Res. 1996, 24, 384. (4) Kawaguchi, H.; Asai, A.; Ohtsuka, Y.; Watanabe, H.; Wada, T.; Handa, H. Nucleic Acids Res. 1989, 17, 6229. (5) Elaı¨ssari, A.; Cros, P.; Pichot, C.; Laurent, V.; Mandrand, B. Colloids Surf. 1994, 83, 25. (6) Lunderberg, J.; Wahlberg, J.; Uhlen, M. Gene. Anal. Technol. 1990, 7, 49. (7) Vener, T. I.; Turchinsky, M. F.; Knorre, V. D.; Yu. V.; Shcherbo, S. N.; Zubov, V. P.; Sverdlov, E. D. Anal. Biochem. 1991, 198, 308.

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styrene latex particles,5,8,9 several features were already clearly emphasized. Adsorbed amounts of ODNs were strongly dependent upon pH and not significantly upon ionic strength, at least in the low pH region. In addition, there was no effect of the nature of the base nor the chain length (if expressed in unit mass m-2) on the maximal adsorbed amount. It was concluded that ODN adsorption was mainly governed by electrostatic attractions; however the influence of nonelectrostatic forces also played a role, a phenomenon which has been confirmed by showing a non-negligible (ODN) adsorption onto negatively charged latex particles.5 Such a behavior has also been reported by Walker et al.10-12 with similar ODNs and cationic latex particles; however in the case of anionic latexes, they showed that the adsorbed amount of ODNs increased in the high ionic strength domain. These results led to the main conclusion that ODNs were mostly adsorbed in a flat monolayer conformation, assuming that each oligonucleotide exhibited a cylindrical form. Obviously, such a conformation is not at all favorable for further hybridization13,14 with complementary sequences. Therefore, covalent coupling with aminoterminated spacer containing ODNs should offer one suitable alternative, provided experimental conditions be optimized for favoring stretched conformation. This method often implies drastic experimental conditions (especially basic pH) for the chemical reaction to be performed, and surfactant should be added to ensure the colloidal stability of the latex particles. Nonionic surfactants are usually preferred, especially poly(ethylene oxide)-based ones due to their tolerance with biologically(8) Elaı¨ssari, A.; Pichot, C.; Delair, T.; Cros, P.; Kurfu¨rst, R. Langmuir 1995, 11, 1261. (9) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C.; Laayoun, A.; Cros, P. Langmuir 1997, 13, 701. (10) Walker, H. W.; Grant, S. B. Langmuir 1995, 11, 3772. (11) Walker, H. W.; Grant, S. B. Langmuir 1996, 12, 3151. (12) Walker, H. W.; Grant, S. B. J. Colloid Interface Sci. 1996, 179, 552. (13) Wolf, S. T.; Haines, L.; Fisch, J.; Kremsky, J. N.; Dougherty, J. P.; Jacobs, K. Nucleic Acids Res. 1987, 15, 2911. (14) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679.

© 1997 American Chemical Society

7022 Langmuir, Vol. 13, No. 26, 1997 Chart 1. Structure of Triton X-405 Surfactant with an Average n Value of 40 as given by Sigma (a) and Poly(thymidylic acid) Nucleotide dT35 Bearing an Aliphatic Primary Amine group (b)

active molecules, particularly enzymes. High HLB (hydrophilic and lipophilic balance) nonionic surfactants (Triton series) are commonly employed for providing efficient steric stability. However, complex interfacial phenomena may result from the adsorption of surfaceactive agents, which could affect subsequent adsorption of other interacting species, such as ODNs in this case. As a preliminary work to perform covalent binding of ODNs onto amino-containing polystyrene latexes, the purpose of this paper is to report on the influence of a nonionic surfactant (Triton X-405) on the adsorption behavior of the ODNs onto these cationic latexes. At first, the adsorption of the emulsifier onto the latexes is precisely examined. Then, the adsorption of a model oligodeoxyribonucleotide (dT35) is studied onto bare and precoated latex particles with a special attention to the adsorption kinetics and isotherms, exchange processes, and desorption. II. Experimental Section Materials. The following materials and reagents were used: Water was of Milli-Q grade (Milli-pore SA, France) and potassium hydrogenophosphate (K2HPO4 and KH2PO4) from Merck was used as a buffer. Triton X-405 [p-(1,1,3,3-tetramethylbutyl)phenoxypoly(ethylene glycol)] with an average of 40 oxyethylene units according to the supplier (Sigma) (Chart 1), was used as received. The oligonucleotide (dT35), bearing an amine group (2HN(CH2)6-) at the 5′ position (Chart 1), was prepared using the standard phosphoramidite chemistry15 on an automatic RNA/ DNA synthesizer (from Applied Biosystems). The protocol was described elsewhere.8 The final product was isolated and purified by high-performance liquid chromatography (HPLC) (Beckmann ODS 10 mm × 25 mm). The molecular weight of the obtained dT35 was Mw ) 10 735 g mol-1 (taking into account the amino link spacer). The oligodeoxyribonucleotide was labeled using the 32P method described elsewhere.16 The 32P-kit was from Boehringer. The concentration of labeled oligodeoxyribonucle(15) Gait, M. J. Oligonucleotides Synthesis, a practical approach; IRL Press: Oxford, 1984. (16) Chang, L. M. S.; Bollum, T. J. J. Biol. Chem. 1971, 246, 909.

Ganachaud et al. otide (32P-dT35) was determined using a TRI-CARB, Model 1900 TR (Packard Instruments). The cationic latex was produced by emulsion-free polymerization of styrene and vinylbenzylamine hydrochloride (VBAH). Details of latex synthesis and characteristics have already been reported.9,17 The mean number-average diameter Dn ) 242 nm and the polydispersity index of 1.005 were obtained from transmission electron microscopy. As determined by colorimetric and fluorescence titrations,17 the surface charge density of the particles originating principally from the amino group of the functional monomer was 17.45 µC cm-2. Methods. Mass Spectrometry Analysis. Positive fast atom bombardment (FAB+) spectra were recorded by the Service Central d’Analyses, Solaize (France), on a ZAB 2-SEQ mass spectrometer from VG micromass equipped with a Cs ion gun and using thioglycerol as a matrix. Critical Micelle Concentration. The critical micelle concentration (cmc) of Triton X-405 was determined in Milli-Q water using either Roos (UV)18 or fluorescence19 methods. (i) The fluorescence method was based on the variation of the fluorescence intensity of surfactant/pyrene mixture solution as a function of surfactant concentration. The fluorescence intensity was measured for an excitation wavelength, λEX ) 300 nm, and an emission wavelength, λEM ) 454 nm, as a function of surfactant concentration. The cmc value was reached when a marked change in the slope of the intensity vs Triton concentration was observed. (ii) The UV method was applied using iodine/surfactant solution. The iodine solution in the absence of surfactant gave a maximum of optical absorption at 450 nm, whereas the addition of surfactant provided a maximum of optical density at 360 nm induced by the formation of a complex between iodine and Triton X-405. For low surfactant concentrations, the optical density was directly related to the surfactant concentration until the micelles formation was attained. Critical Coagulation Concentration of Latex Particles. The critical coagulation concentration (ccc) was determined by measuring the time dependence of the optical density of diluted latex after addition of a given concentration of sodium chloride.20-22 The optical density (OD) was measured at 580 nm wavelength using a UVIKON 930 spectrophotometer. The stability factor W is given by the ratio of two initial slopes of OD versus time (dOD/dt from the tangent of the OD curve in the early stages)

W ) (dOD/dt)r/(dOD/dt)s where r and s mean rapid and slow aggregation respectively. Electrophoretic Mobility of Latex Particles. The electrophoretic mobility23,24 of latex particles was determined using a Zeta Sizer 3 (from Malvern Instruments) at 20 °C. The measurements were carried out with latex samples highly diluted in 10-3 M NaCl, and the pH was controlled by adding either HCl or NaOH. Each point is the average of at least three measurements. Capillary Electrophoresis. Capillary electrophoresis was performed using an Applied Biosystems instrument equipped with a silica capillary (72 cm length and 50 µm as the internal diameter). Samples were introduced into the capillary by a hydrodynamic injection for 10 s at 40 °C. The optical density was measured at a 260 nm wavelength in carbonate buffer (20 mM, pH 9.3). Adsorption of Triton X-405 onto Aminated Latex Particles. All the adsorption and desorption experiments were performed in 2 mL Eppendorf tubes. To determine the adsorbed amount of Triton X-405 on the latex particles, the conventional (17) Ganachaud, F.; Mouterde, G.; Delair, T.; Elaı¨ssari,A.; Pichot, C. Polym. Adv. Technol. 1994, 6, 480. (18) Roos, S.; Olivier, J. P. J. Phys. Chem. 1959, 63, 1671. (19) Cochin, D.; Zana, R.; Candau, F. Polym. Int. 1993, 30, 491. (20) Bensley, C. N.; Hunter, R. J. J. Colloid Interface Sci. 1982, 88, 546. (21) Overbeek, J. T. G. Adv. Colloid Interface Sci. 1982, 16, 17. (22) Kitahara, A.; Ushiyama, H. J. Colloid Interface Sci. 1973, 43, 73. (23) Hunter, R. J. Zeta Potential in Colloid Science, Principles and Applications; Academic Press: London, 1981. (24) Kitahara, A.; Watanabe, A. In Electrical Phenomena at interfaces, Fundamental Measurements, and Applications; Marcel Dekker: New York, 1984.

Cationic Latexes, Triton X-450, Oligonucleotide, Adsorption depletion technique5,8,9 was performed. Initially, a given amount of Triton X-405 dissolved in phosphate buffer (10-3 M at pH 6) is added to 50 µg of latex particles and completed with phosphate buffer in order to obtain 1 mL of final volume. After 10 h of incubation time, the bulk Triton X-405 concentration was measured by a UV spectrophotometer at 275 nm. The adsorbed amount was then deduced after latex centrifugation and analysis of the supernatant. Desorption of Triton X-405. The desorption of Triton X-405 was carried out as a function of washing steps. Precoated latex with high amounts of adsorbed Triton X-405 was washed using Milli-Q water and the residual adsorbed amount was determined from supernatant analysis. This washing step (centrifugationremoval-redispersion-centrifugation) was repeated until the residual adsorbed amount of Triton remained constant. Adsorption of Poly(thymidylic acid) to Aminated Latex Particles. The adsorption of dT35 onto bare and precoated latex particles was also investigated using the same depletion method as for the surfactant adsorption. The oligonucleotide was diluted in phosphate buffer at a given pH and ionic strength (controlled by adding NaCl). After 2 h8,9 of adsorption at room temperature, the supernatant was analyzed after centrifugation from which the adsorbed amount was determined at a given pH and ionic strength. Capillary electrophoresis was used as an analytical technique. Poly(thymidylic acid) concentrations were determined from a calibration curve. Desorption of Poly(thymidylic acid). In all desorption experiments, the latex particles bearing 1 mg m-2 dT35 were prepared at pH 5, 10-3 M ionic strength in phosphate buffer and using bare latex particles. Desorption of dT35 as a function of washing steps8 was investigated by replacing only 25% of the supernatant with the same volume of a buffer solution at a given pH and ionic strength (free or containing 1% Triton X-405 concentration). The desorption process was allowed to stand overnight, and the residual adsorbed amount of dT35 was determined by measuring the difference between the initial adsorbed one and the free oligonucleotides in the medium. The effect of pH and ionic strength on the desorption of poly(thymidylic acid) was investigated using buffer at a given pH and ionic strength and containing 1% Triton X-405. The residual adsorbed amount was determined after two washes for each sample and one night incubation time between each wash. Exchange Kinetics of Poly(thymidylic acid). The exchange experiments were performed at basic pH 9.2 both on the bare and precoated Triton X-405 latex particles. To study the exchange rate of adsorbed oligodeoxyribonucleotide as a function of time, two latexes were prepared at saturation conditions using labeled 32P-dT35 and nonlabeled dT35 respectively. After adsorption equilibrium, the adsorbed amount of oligodeoxyribonucleotide was first determined, then the supernatant corresponding to labeled dT35 was exchanged with that of the nonlabeled one in order to maintain the adsorption equilibrium. The desorbed amount of 32P-dT35 was then measured (by analyzing the bulk solution after centrifugation) and related to the oligodeoxyribonucleotide exchanged amount as a function of time. Such a method has been used by several authors in order to investigate the protein exchange rate25 and the dynamic stability of the adsorbed polyelectrolytes layer.26-28

III. Results and Discussion The cationic latex was cleaned by repetitive centrifugation in order to remove all residual electrolytes and polyelectrolytes. The degree of cleanliness was determined by measuring the conductivity of the supernatant as a function of washing steps using pure water (Milli-Q water). Before the effect of Triton X-405 on the adsorption of single-stranded poly(thymidylic acid) (dT35) onto latex particles was studied, adsorption and desorption of (25) Ball, V.; Huetz, P.; Elaı¨ssari, A.; Cazenave, J. P.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. 1994, 91, 7330. (26) Pefferkorn, E.; Carroy, A.; Varoqui, R. J. Polym. Sci. 1985, 23, 1997. (27) Pefferkorn, E.; Haouam, A.; Varoqui, R. Macromolecules 1989, 18, 2252. (28) Pefferkorn, E.; Carroy, A.; Varoqui, R. Macromolecules 1985, 22, 2677.

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Figure 1. FAB+ mass spectroscopy of Triton X-405. Two broad distributions of peaks (% of Triton molecules) are observed, one centered on m/z ) 1545 (single ionized molecules), the other one on 690 g mol-1 (twice ionized molecules). The close coupled peaks also observed correspond to H+ and Na+ ionized-like molecules.

this surfactant have been first examined. Such a preliminary work was necessary in order to better understand the particle surface modification originated by the presence of the surfactant and its effect on the ODN adsorption. A. Adsorption Behavior of Triton X-405. A.1. Triton X-405 Characterization. Molecular characterization of the Triton surfactant has been completed using FAB+ mass spectroscopy measurements, as shown in Figure 1. Two peak distributions are clearly seen on this spectrum, which corresponds to Triton molecules ionized with one proton ([M + H]+ distribution, centered on m/z ) 1545 g mol-1) and two protons ([M + 2H]2+ distribution, with a maximum at about 690 g mol-1). Furthermore, two very close peaks were observed, corresponding to the Triton molecules bearing either H+ or Na+ (weight difference of 22 g mol-1 between the two peaks). Then, the Triton is almost pure, but with a broad distribution of the poly(ethylene oxide) (PEO) length (starting from 20 to 45 EO units and with a maximum centered at 30 EO). The observed distribution suggests the presence of various surface-active species covering all the HLB (hydrophilic and lipophilic balance) spectrum. The molecular weight used in this study was Mw ) 1545 g mol-1. The critical micelle concentration (cmc) of Triton X-405 was determined in Milli-Q water using the two methods described in the experimental part; UV and fluorescence methods. In this latter case, the cmc was found to be 1.3 mmol L-1. Using the UV method, a value slightly lower (0.95 mmol L-1) is obtained which can be explained by the lack of sensitivity of this method. A.2. Adsorption Isotherm of Triton X-405 onto Latex Particles. The adsorption isotherm of Triton X-405 onto cationic latex particles was first established at pH 6 and 10-3 M ionic strength of NaCl as reported in Figure 2 in which the adsorbed amount of Triton X-405 Ns (mg m-2) is plotted against the bulk concentration Ceq (mg mL-1). The adsorption isotherm appears to follow a Langmuirian type model with a plateau value attained for 0.01 mg mL-1 Triton X-405 of equilibrium concentration. According to the maximal adsorbed amount, 0.5 mg m-2, an average molecular surface area of Triton X-405 of 513 Å2 was estimated. This value is indeed larger than

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Figure 2. Adsorption isotherm of Triton X-405 onto cationic latex particles (at 20 °C, pH 6, and 10-3 M ionic strength) after 10 h of adsorption.

the one reported29 for sulfate-charged polystyrene latex particles, i.e., 347 Å2. Such a discrepancy may reflect a significant difference in the interactions between the surfactant and the water-polymer interface. As pointed out by Zhao et al,30 the adsorption of poly(ethylene oxide)based surfactants onto polystyrene latexes is somewhat complex. In addition to the hydrophobic interactions originating from the hydrocarbon tails, hydrogen bonding between nonprotonated amino groups and the ether oxygens of the poly(ethylene oxide) units may also take place. This later point was not observed in our case, since the maximal adsorbed amount of Triton X-405 was found to be pH independent. The difference between the reported29 surface area and the obtained one can also be attributed to the molecular weight of Triton X-405 used (40 EO units instead of 30). The adsorption of low molecular weight surfactants onto latex particles can be usually interpreted by a Langmuirtype model as proposed by several authors.31-35 By using Langmuir representation, the affinity constant (K ) 2.45 × 103 L mol-1) and the maximal adsorbed amount (Ns,∞ ) 0.48 mg m-2) were determined. The affinity of the PEO-containing surfactants with polystyrene latex surfaces is strongly dependent on the PEO chain length as pointed out by Kronberg et al.;36 the longer the chain the lower the affinity. It is usually considered that the hydrophobic parts of the surfactants are mostly involved in the adsorption onto the hydrophobic particles surfaces.36-38 The maximal adsorbed amount of Triton X-405 indicates that it forms a monolayer coverage on the aminated polystyrene latex particles. The Triton adsorbed amount at 10-3 M ionic strength was found to be pH independent, reflecting that adsorption was principally governed by hydrophobic interactions between the hydrophobic part of the Triton X-405 and the polystyrene surface. Similar results and observations were (29) Romero-Cano, M. S.; De Las Nieves, F. J.; Martin-Rodriguez, A. Unpublished data. (30) Zhao, J.; Brown, W. J. Colloid Interface Sci. 1996, 179, 281. (31) Painter, D.; Hall, D. G.; Wyn-Jones, E. J. Chem. Soc., Faraday Trans 1988, 84, 773. (32) Paxton, T. R. J. Colloid Interface Sci. 1969, 31, 19. (33) Ali, S. I.; Steach, J. C.; Zollars, R. L. Colloids Surf. 1987, 26, 1. (34) Hoeft, C. E.; Zollars, R. L. J. Colloid Interface Sci. 1996, 177, 171. (35) Tuin, G.; Stein, H. N. Langmuir 1995, 11, 1284. (36) Kronberg, B.; Ka¨ll, L.; Stenius, P. J. Dispersion Sci. Technol. 1981, 2, 215. (37) Piirma, I.; Chen, S. R. J. Colloid Interface Sci. 1980, 74, 90. (38) Bo¨hmer, M. Langmuir 1990, 6, 1478.

Ganachaud et al.

Figure 3. Residual adsorbed amount of Triton X-405 onto latex particles as a function of washing steps.

reported by Ahmed et al39 using a Triton X-100/anionic latex system. A.3. Desorption Study. The desorption of preadsorbed surfactant from the latex particles was investigated as a function of washing steps. The latex particles were first saturated by adsorbing the Triton X-405 (0.48 mg m-2 at pH 6 and 10-3 M ionic strength), and then the supernatant was replaced by Milli-Q water (pH 6). The residual amount of adsorbed surfactant versus the number of washing steps is reported in Figure 3. It is worth mentioning that after several washing steps, the residual adsorbed amount of surfactant is not negligible, since the plateau value represents about 20% (0.1 mg m-2) of the initial adsorbed amount (0.5 mg m-2) after five washes. Such a residual adsorption might originate from the strong adsorption of low HLB surfactant species contained in Triton as discussed by Kronberg et al.36 and already observed in the case of similar PEO-containing nonionic surfactants.40 In the following study, the precoated latex particles were prepared under the same desorption conditions and the residual adsorbed amount of Triton X-405 was always the same, on the order of ∼0.1 mg m-2 in all experiments. A.4. Electrokinetic Study. The electrophoretic mobility (µe) of the latex particles was measured as a function of pH, in 10-3 M NaCl solution at 25 °C. Figure 4 shows the electrophoretic mobility of latex particles as a function of pH for bare and precoated latex particles with Triton, respectively. Significant differences are observed according to the type of latexes. At first both latexes exhibited positive µe in the low pH region, which corroborates the presence of labile cationic charges onto latex surfaces. However, the slope of the µe variation vs pH decreases more rapidly in the case of the precoated latex than for the bare one. Consequently, the isoelectric point (IEP) of the former latex appears at lower pH (pH ∼8) than for the latter one (pH ∼10). In the case of bare latex particles, this IEP is indeed very close to the pKa of the VBAH monomer.41 The observed behavior of µe versus pH for precoated latex was attributed to the presence of residual nonionic surfactant onto latex particles, probably inducing a shift in the slipping plane42-47 away from the surface. (39) Ahmed, M. S. Thesis, Lehigh University, Bethlehem, PA, 1981. (40) Emelie, B.; Kong, X. Z.; Pichot, C. Polymer latex II, ed.; Plastics and Rubber Institute: London, Mai 1985, Paper No. 9. (41) Sauzedde, F.; Ganachaud, F.; Elaı¨ssari, A.; Pichot, C. J. Appl. Polym. Sci. 1997, 65, 2331. (42) Suzawa, T.; Shirahama, H. Adv. Colloid Interface Sci. 1991, 35, 139. (43) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344.

Cationic Latexes, Triton X-450, Oligonucleotide, Adsorption

Figure 4. Electrophoretic mobility of bare and precoated latex particles as a function of pH at 25 °C and 10-3 M of NaCl. The precoated latex particles contain 0.1 mg m-2 of residual adsorbed Triton X-405.

Figure 5. Stability factor (W) versus NaCl for bare and precoated latex particles (at pH 6 and 25 °C) in log-log plot. The precoated latex particles contain 0.1 mg m-2 of adsorbed Triton X-405.

This effect induces a decreasing in the electrophoretic mobility and a shift in the isoelectric point (IEP) from 10 to 8. A.5. Colloidal Stability. As illustrated in Figure 5, showing a log-log plot of the variation of the stability factor (W) as a function of ionic strength, the critical coagulation concentration (ccc) for the precoated latex (0.17 M) is lower than that for the bare particles (0.26 M). The destabilization of precoated latex particles induced by high ionic strength (>0.26 M), was presumably a result of a flat conformation of residual adsorbed amount of Triton (0.1 mg m-2 less than monolayer). A similar result has been already reported by Chiming47 using a Triton X-100/ polystyrene latex system. The stability of precoated latex becomes important only when the particles are fully covered with surfactant molecules. In that case, the hydrophilic EO chains are extended into the aqueous phase and providing steric protection to the latex particles as qualitatively observed by adsorbing one monolayer (0.45 mg m-2) of surfactant. More information on the effect of surfactant concentration on the colloidal stability of polystyrene latex/Triton X-100 is reported by Chiming.47 (44) Tamai, H.; Hasegawa, M.; Suzawa, T. J. Appl. Polym. Sci. 1989, 38, 403. (45) Tamai, H.; Fujii, A.; Suzawa, T. J. Colloid Interface Sci. 1987, 116, 37. (46) Ono, O.; Jidia, E. Polym. J. 1975, 7, 363. (47) Chiming, M. Colloids Surf. 1987, 28, 1.

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Figure 6. Percentage of adsorbed oligonucleotide (dT35) as a function of time at 20 °C, 0.1 mg mL-1 of dT35, 0.1 mg mL-1 of latex particles, 10-2 M ionic strength, and pH 5.

B. Effect of Residual Triton onto the Oligonucleotide Adsorption. Several studies have recently been concerned with the physical adsorption of ODNs onto sulfate-5 or amino-charged8,9 bare latexes; however, no systematic work has been investigated on precoated particles in order to examine how the modification of the surface properties may affect the adsorption and desorption processes of these polyelectrolyte species. The adsorption competition between free anionic groups from the phosphate buffer and ODN molecules was neglected in this study. B.1. Adsorption Kinetic of Oligonucleotide. The adsorption behavior of ODNs onto colloid supports can be discussed by analogy with the case of small-size polyelectrolytes. The adsorption kinetics of polyelectrolytes onto colloidal supports were largely studied and discussed in terms of mechanistic process as reported in the literature.48,49 The adsorbed amount of oligonucleotide onto latex particles was found to be time-dependent as recently pointed out.8,9 The adsorption kinetics process was affected by the surface nature of the latex particles as illustrated in Figure 6, in which the percentage of adsorbed amount of ODN was represented versus time. The maximal adsorbed amount of ODN onto bare latex particles was reached more rapidly than in the case of precoated surface. The difference on the adsorption kinetics of ODN onto precoated latex can be attributed to the effect of residual Triton X-405 onto the latex particles. This induces not only a decrease in the amplitude of the attractive electrostatic forces but also reduction of available free surface and possible exchange between free ODN and adsorbed surfactant. All these phenomena contribute to the slow adsorption step of ODN onto the precoated latex by increasing the adsorption barrier. B.2. Adsorption Isotherms of Oligonucleotide. Adsorption isotherms of ODN onto coated latex particles at 10-2 M ionic strength were carried out as a function of pH in order to point out the effect of residual adsorbed Triton (0.1 mg m-2) on the adsorption of ODN. As depicted in Figure 7, the adsorption isotherm of dT35 was established onto precoated latex particles. At acidic pH, the adsorption isotherms are of high affinity in both cases, and when the pH increases the affinity decreases as reflected by the slope of the adsorption isotherm for low ODN equilibrium concentration. All the adsorption (48) Pefferkorn, E.; Jean-Chronberg, A. C.; Varoqui, R. C. R. Acad. Sci. Paris, 1989, 308, 1203. (49) Fleer, G. J.; Leklema, J. Makrom. Chem., Macromol. Symp. 1988, 17, 39.

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Figure 7. Adsorption isotherms of oligonucleotide onto precoated latex particles. Samples were mixed and incubated for 2 h at 20 °C, 10-2 M ionic strength at a given pH.

Figure 8. Effect of pH on the maximal adsorbed amount of dT35 onto bare and precoated latex particles as a function of pH at 20 °C and 10-3 M ionic strength.

isotherms reach a plateau value at solution concentration of 2 nmol mL-1 of ODN. The adsorption isotherms of dT35 show higher affinity and larger adsorbed amount for bare latex particles as reported in reference9 than for precoated one. These results were attributed to the surface modification that originated from the residual nonionic surfactant molecules. B.3. Effect of pH on the Oligonucleotide Adsorption. As pointed out in previous works,5,8,9 the maximal adsorbed amount of single-stranded DNA fragments onto bare cationic latex particles drastically decreases upon increasing the pH. This dependence was principally attributed to the surface charge modification induced by changing the pH of the medium from acidic to basic. As a result, the adsorption of such polyelectrolyte molecules was principally discussed in terms of electrostatic forces with a small contribution of hydrophobic interactions. Figure 8 shows the maximal adsorbed amount of ODN onto bare and precoated latex particles as a function of pH. The adsorbed amount of oligonucleotide was found to decrease when the pH increases, and in all cases, two slopes were observed. In the first part corresponding to low pH domain, the adsorbed amount linearly decreases up to a pH near the isoelectric point of the bare and precoated latex particles, respectively. This indicates that electrostatic interactions act as an important driving force for the adsorption of ODN in this pH domain. The second part of the slope shows a dramatic decrease of the adsorbed amount of ODN onto precoated latex compared to the bare one. In the case of bare latex particles, the decrease in

Ganachaud et al.

Figure 9. Effect of ionic strength on the maximal adsorbed amount of dT35 onto precoated latex particles as a function of pH.

the adsorbed amount (from 0.5 to 0.2 mg m-2) in the pH range from pH 9 to 10.5 was attributed to a larger decrease of the adsorbed amount induced by electrostatic interactions, because attractive forces became less and less important. In the case of precoated latex particles, the adsorbed amount of ODN decreases from 0.7 to practically 0 mg m-2 as the pH increases from pH 7 to 10.5. The absence of adsorption at higher pH would be caused by the presence of the PEO layer on the particle, which prevents any adsorption via hydrophobic interactions. In addition, the presence of interfacial surfactant can also induce a very extended Debye length which reduces the adsorption of ODN in this pH range. B.4. Effect of Ionic Strength on the Oligonucleotide Adsorption. Considering the strong influence of salt concentration on the adsorption of polyelectrolytes onto solid-phase supports,50-52 it was of interest to examine how the ionic strength variation could also affect the ODN adsorption onto these latex particles. This study was only investigated in a range of ionic strength between 10-3 and 10-1 M due to the limited colloidal stability of the latex particles above 10-1 M. Figure 9 shows the effect of ionic strength on the maximal adsorbed amount of dT35 onto bare and precoated latex particles. In the case of the bare particles, as previously reported,5 there is no significant effect of the ionic strength on the adsorption of ODNs (at least in the investigated range of ionic strength). However, in the case of precoated particles, a slight decrease of the adsorbed amount of dT35 is observed upon increasing the ionic strength. Such a behavior may be attributed to the surface modification of the particles induced by the residual adsorbed amount of Triton X-405, making the interface more hydrophilic and reducing hydrophobic adsorption of ODN. In addition, the presence of residual interfacial surfactant can also affect the Debye length even if the adsorbed amount of Triton was too low (∼0.1 mg m-2). The presence of adsorbed surfactant increases the Debye length thickness compared to the bare latex, resulting in a decrease of the ODN adsorption for a given ionic strength. B.5. Exchange Processes in the Adsorption of Oligonucleotide. The adsorption of polyelectrolytes onto opposite-charged surfaces has been largely investigated as reported in the literature. In most cases, the adsorbed layer was supposed to be at equilibrium state. However, (50) Hoogeveen, N.; Cohen Stuart, M. A.; Fleer, G. J. J. Colloid Interface Sci. 1996, 182, 133. (51) Hesselink, F. T. J. Colloid Interface Sci. 1977, 60, 448. (52) Melzak, K. A.; Sherwood, C. S.; Turner, R. F. B.; Haynes, C. A. J. Colloid Interface Sci. 1996, 181, 635.

Cationic Latexes, Triton X-450, Oligonucleotide, Adsorption

the presumed adsorption equilibrium was found to be dependent on the experimental conditions (temperature, bulk concentration, solvent, pH, and ionic strength); the modification of one of these parameters leads to a new equilibrium situation. Therefore, the reversibility of the polyelectrolyte adsorption onto a given support was principally discussed in terms of exchange process: (i) exchange between adsorbed polyelectrolytes and solvent molecules; (ii) exchange between small adsorbed polyelectrolytes and longer ones (in the case of polydisperse sample); (iii) rearrangement of adsorbed layer. The interfacial exchange established between the adsorbed layer of macromolecules (polyelectrolytes, proteins and DNA) and the free molecules in the solution involved a dynamic equilibrium, between the two phases. A few papers dealt with the interfacial exchange.25-27,53,54 However, there is no systematic study of such a process. The theoretical aspect of exchange phenomenon was studied by de Gennes55 who pointed out that the real exchange process was principally governed by the bulk concentration. Experimental results obtained by Pefferkorn et al.26,27 correlated well the prediction of this theory.55 The exchange rate was also found to be dependent on conditions under which the adsorption and the exchange were performed as pointed out by Ball et al.25 in the case of the protein/latex system and on the molecular weight of protein solutions as reported by Vroman et al.56,57 The interfaces prepared by incubating the support in a very diluted solution of polyelectrolytes were much more stable than those prepared in a very concentrated solution of macromolecules.26 This phenomenon was assigned to the reconformation “relaxation” process of adsorbed macromolecules at solid/liquid interfaces.58 To investigate the exchange phenomenon between adsorbed radiolabeled dT35 and nonlabeled dT35 free in the bulk solution, the mass transfer from the surface to the solution was determined by measuring the concentration of labeled free molecules in the medium. Figure 12 shows the amount of exchanged labeled dT35 versus time. The exchanged amount was calculated from the amount of labeled ODN molecules released from the particles during the incubation time. The bulk dT35 concentration measured during this process was found to be constant. This indicates that the residual adsorbed amount of labeled and unlabeled dT35 remained constant (∼0.4 mg m-2) and that the increasing of the radioactivity of the bulk solution was only due to the exchange process. As illustrated in Figure 10, the exchange was slow and monotonous irrespective of the nature of the latex surface. The exchange rate in these adsorption equilibrium conditions deduced from the initial slope of the curve was found to be 1.3 µg m-2 h-1 in both cases (bare and precoated latex). Such a result explains that the duration stay of labeled ODNs in the adsorbed layer was higher compared to the equilibrium time (100 min on the bare latex particles and more than 200 min on the precoated one as shown in Figure 6). The Triton X-405 is found to affect the adsorption exchange, although the exchanged amount seems to be slightly higher in the case of precoated latex as shown in Figure 10. This can be attributed not only to the ODNODN exchange but also to the displacement of a small (53) Carroy, A. Ph.D. Thesis, Universite´ Louis Pasteur, Strasbourg, France, 1986. (54) Haoum, A. Ph.D. Thesis, Universite´ Louis Pasteur, Strasbourg, France, 1986. (55) de Gennes, P. G. C. R. Acad. Sci. Paris 1985, 20, 1399. (56) Vroman, L.; Adams, A. L.; Klings, M. Fed. Proc. 1971, 30, 1494. (57) Vroman, L.; Adams, A. L. J. Colloid Interface Sci. 1986, 111, 391. (58) Varoqui, R.; Pefferkorn, E. Makrom. Chem., Macromol. Symp. 1988, 17, 87.

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Figure 10. Amount of dT35 exchanged as a function of time. The adsorbed amounts were 0.5 and 0.3 mg m-2 onto bare and precoated latex, respectively (at 20 °C, pH ) 9.2 and 10-2 M ionic strength). The exchange was performed using surfactantfree buffer.

part of the adsorbed surfactant molecules by the ODNs (Triton-ODN exchange). As a conclusion, the observed exchange phenomenon between adsorbed and free ODNs indicates that the adsorption of such nucleic acids onto bare cationic polystyrene latex is a slightly reversible adsorption process; however, the ODN exchange on the precoated latex is somewhat complex, since the exchange between adsorbed surfactant and free ODNs should be taken into account. C. Desorption Study of Adsorbed Oligonucleotide. In order to further perform the covalent immobilization of ODN molecules onto the latex particles, it is important to get free of the molecules which could be simultaneously adsorbed. Therefore, in the last part of this paper, desorption study of preadsorbed ODNs onto latex particles was investigated as a function of the pertinent parameters which were found to play a role in the adsorption process: the presence of Triton, pH, and ionic strength. It was already shown in a previous paper8 that desorption of ODNs from cationic particles could be induced through the replacement of the supernatant of the latex at adsorption equilibrium by a buffer solution allowing control of the pH. The higher the pH of the substituted serum, the larger the amount of ODN which could be desorbed. The residual adsorbed amount of ODN, after desorption at a given pH, was found to be similar to that corresponding to the adsorption preformed at this pH. Two main reasons were proposed for explaining the desorption process: (i) the dilution effect of the bulk ODN concentration affecting the adsorption equilibrium; (ii) a reduction of the surface charge density by increasing pH from acidic to basic conditions resulting in a decrease of electrostatic interactions between the ODNs and the latex. On the basis of these observations, the desorption study was conducted with two requirements: (i) to find out optimal conditions ensuring complete desorption of ODNs; (ii) to carry out the desorption experiments in the presence of Triton, since as already mentioned this surfactant is generally used in covalent binding recipes. For the sake of simplicity, the desorption behavior of ODN was considered in the case of bare cationic latex particles on which dT35 was adsorbed (at pH 5.0, 10-2 M ionic strength) providing 1.0 mg m-2 adsorbed amount. C.1. Effect of Triton on the Desorption of Oligonucleotide. A first study was directed with a view to examining the influence of Triton when present in the

7028 Langmuir, Vol. 13, No. 26, 1997

Figure 11. Residual adsorbed amount of dT35 onto bare and precoated latex particles as a function of washing steps. Adsorption was performed onto bare latex particles at pH 5 and 10-2 M ionic strength.

buffer solution. The following strategy was defined in order to analyze the desorption induced by small changes in the supernatant composition for each washing step: 25% of the supernatant was replaced by a borate buffer solution (pH 9.25, ionic strength 10-2 M) containing or not the nonionic surfactant (1% (w/w) Triton X-405). As illustrated in Figure 11, reporting the residual adsorbed ODN amount (Ns) as a function of washing steps, it is clear that the desorption of dT35 is more marked when using buffer containing Triton than with surfactant-free buffer. In both cases, the desorption of ODN mostly occurs during the two first washes because of the pH shift from pH 5 to 9.25 (reduction of electrostatic interactions). After the third wash, the desorbed amount of dT35 becomes small until it reaches a plateau. Concerning the effect of Triton, it may be assumed, due to an exchange process, that a part of ODN is displaced by nonionic surfactant molecules resulting in a larger ODN desorption in the two first washes until all weakly adsorbed ODN are desorbed. On the basis of this preliminary study, the desorption was further investigated upon using buffer containing 1% Triton X-405. C.2. The Effect of pH on the Desorption of Oligonucleotide. In that case, washing conditions were changed in order to reduce maximal ODN desorption. With the same bare latex particles bearing 1.0 mg m-2 of ODN (prepared at pH 5 and 10-2 M ionic strength), the whole supernatant was replaced by a buffer solution containing Triton X-405 and the residual adsorbed amount of ODN at a given pH was determined after two washes. As shown in Figure 12, which reports the residual adsorbed amount (Ns) versus pH of the dispersed medium, the amount of desorbed ODN increases upon increasing pH of the medium. It is indeed found that 20% of ODN is desorbed at pH 8 instead of 60% at pH 11. As already mentioned, this desorption process is caused by a reduction of the surface charge density upon increasing the pH; this was indeed reflected by the electrophoretic mobility measurements (Figure 4). Such a decrease in the cationic surface charges results in a corresponding reduction of the attractive forces between negatively charged ODN and positively charged latex particles. In addition, due to the exchange phenomena between adsorbed ODNs and free Triton molecules, this desorption is more important in the presence of the surfactant. In that case, it was also shown a more important decrease of the electrophoretic mobility of the precoated latex particles, due to a screening effect induced by the adsorbed surfactant. C.3. The Effect of Ionic Strength on the Desorption of Oligonucleotide. As illustrated in Figure 13,

Ganachaud et al.

Figure 12. Residual adsorbed amount of dT35 onto bare latex particles as a function of pH. Adsorption was performed onto bare latex particles. The desorption was carried out after two washes using buffer (at a given pH, 10-2 M ionic strength, and 1% Triton X-405).

Figure 13. Residual adsorbed amount of dT35 onto bare latex particles as a function of ionic strength. Adsorption was performed onto bare latex particles with Ns ) 1.0 mg m-2 at pH 5 and 10-2 M ionic strength. The desorption was carried out after two washes using buffer (at various ionic strengths, pH 10, and 1% Triton X-405).

the desorption of ODN seems to occur relatively slightly for ionic strength ranging between 10-2 and 10-1 M and dramatically increases for higher ionic strength. The residual adsorbed ODN amount is indeed practically nil at 5 × 10-1 M ca. (0.05 mg m-2). This effect can also be explained by a reduction of the attractive forces between ODNs and surface charges on the latex induced by both a basic pH and higher ionic strength. Moreover, the influence of the surfactant cannot be discarded as already discussed. Residual Triton molecules make the particle surface more hydrophilic causing a decrease of the interactions with ODNs. It may be added that ionic strength (in the investigated range) would not have any significant effect on the adsorption of nonionic surfactant molecules contrary to the case of charged species (ODNs). As a conclusion, this study shows that it is possible to control the desorption process of adsorbed ODNs onto cationic latex particles, upon modification of both pH and ionic strength of the dispersed medium. The presence of the nonionic surfactant also plays a predominant role on this process through a cooperative effect. IV. Conclusion The adsorption of single-stranded DNA fragments (ODN) has been studied at room temperature onto cationic

Cationic Latexes, Triton X-450, Oligonucleotide, Adsorption

amino-containing polystyrene latex particles being or not preadsorbed emulsifier molecules (Triton X-405). A preliminary study was devoted to the adsorption behavior of the nonionic emulsifier onto this latex then the adsorption of poly(thymidylic acid) onto bare and precoated latex particles was considered. The adsorption of Triton X-405 onto cationic latex particles was found to follow a Langmuirian type with an affinity constant K ) 2.45 × 103 L mol-1 and a maximal adsorbed amount Ns,∞ ) 0.48 mg m-2. The adsorption of this nonionic surfactant was not pH dependent, which suggested that hydrophobic interactions mostly governed the process. It was also found that about 20% of the total adsorbed Triton X-405 remained physically adsorbed onto the particles even after an extensive cleaning procedure. The presence of this residual surfactant was indirectly evidenced through the modification of the electrokinetic properties and colloidal stability of the latexes compared to that of the bare particles. Secondly, the adsorption and desorption behaviors of dT35 were examined with bare and precoated latex particles, and the main results were as follows: The presence of Triton X-405 molecules was found to significantly affect both the kinetics and adsorption isotherms of the ODNs onto such latex particles. The adsorption plateau was reached less rapidly for precoated particles and its value was lower than that for bare ones. This was attributed to the modification of the particle surfaces caused by the residual adsorbed Triton, inducing high adsorption barrier and excluded surface area. This effect was also clearly evidenced through the adsorption behavior of ODNs as a function of pH and ionic strength. It was worthy to mention that nonionic surfactant molecules totally reduced the hydrophobic inter-

Langmuir, Vol. 13, No. 26, 1997 7029

actions between ODNs and bare particles, especially at basic pH. Varying the ionic strength in the range 10-4 10-1 M slightly affected the ODNs adsorption, but only in the case of precoated particles. The exchange process between adsorbed and free ODNs was also studied, leading to an exchange rate around 1.3 µg m-2 h-1 for both latexes, indicating that the adsorption of ODNs onto cationic polystyrene latex particles was a reversible process. In addition, the exchange in the case of precoated latex particles was not only ODN-ODN exchange but also Triton-ODN as well. Finally, the desorption behavior of the ODNs from these latexes was investigated. It was first pointed out that the desorption was more efficient when using buffer containing 1% Triton X-405 than without. In addition, carrying out the desorption above pH 10 and at high ionic strength made the desorption of ODNs almost complete. From this study, it is clear that the presence of preadsorbed Triton X-405 drastically affects the interactions between ODNs and cationic latex particles. One interesting feature is that it contributes to reduce the physical adsorption of ODN, which is quite suitable for further performing the covalent immobilization of ODN onto these amino-containing latexes. Acknowledgment. The authors are indebted to Dr. E. Pefferkorn (ICS, Strasbourg, France) and Dr. T. Delair, P. Cros, and A. Laayoun (Bio-Me´rieux, Lyon, France) for critical reading and helpful discussions and suggestions. We are also grateful to N. Ferraton, C. Brun, and V. Thomet for the preparation of oligonucleotides and V. Balladur for radioactivity measurements. LA9704819