Structure of Adsorbed and Grafted Single-Stranded DNA Fragments

A small angle neutron scattering study of the interface between solids and oil-continuous emulsions and oil-based microemulsions. Philip A Reynolds , ...
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Langmuir 2000, 16, 1261-1269

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Structure of Adsorbed and Grafted Single-Stranded DNA Fragments on Aminated Latex Particles: A Small-Angle Neutron Scattering Study Abdelhamid Elaı¨ssari,*,† Yves Chevalier,‡ Franc¸ ois Ganachaud,† Thierry Delair,† and Christian Pichot† UMR-103 CNRS-bioMe´ rieux, ENS de Lyon, 46 Alle´ e d’Italie, 69364 Lyon, Cedex 07, France, and LMOPS-CNRS, BP 24, 69390 Vernaison, France Received June 3, 1999. In Final Form: September 24, 1999 Adsorption and covalent binding of polythymidylic acid (dT35) on aminated polystyrene latex particles have been studied. Both the electrostatic interactions between negative charges of the dT35 and the positive charges of the cationic latex particles and hydrophobic interactions are the driving forces for the adsorption. Small-angle neutron scattering (SANS) has been used to determine the structure of the adsorbed or grafted dT35 layers. The thickness of the adsorbed oligonucleotide layer on the latex particles is small with respect to the extended length of dT35 molecules (∼120 Å) indicating that the adsorbed molecules lay close in a flat conformation on the surface, irrespective of the pH and ionic strength. In contrast, the covalently grafted dT35 molecules at basic pH and high surface coverage extends more radially into the solvent, giving rise to a thicker layer in comparison to the case of adsorption.

Introduction Adsorption of nucleic acids onto various solid supports is, nowadays, of great interest in the field of molecular biology as well as in diagnostics and medicine. These widespread applications could not be undertaken or even envisaged without highly efficient automated oligonucleotide synthesis tools. These syntheses, which are currently achieved by using phosphoramidite chemistry,1 provide large amounts of oligodeoxynucleotides (ODNs), allowing their use in diagnostic assays based on nucleic acids hybridization.2,3 Sandwich-type assays, which require multiple discrete sequences available for hybridization of their complementary target DNA or RNA, are based on oligonucleotide capture sequences grafted onto various solid supports.4 One drawback of this nucleic acid immobilization method is that both grafted and adsorbed oligonucleotides coexist at the surface, and the adsorbed molecules can be desorbed from the support during storage. Moreover, hybridization rates are slower than for the corresponding reaction in a bulk solution, and this recognition process may even become inefficient at solidliquid interface. This effect can be attributed to the poor availability of nucleic acids for hybridization when the reaction takes place at a surface. Such situations may be encountered in cases where the bases that are responsible for the chemical recognition phenomenon are oriented toward the solid support or buried in the oligonucleotide layer instead of protruding toward the analyte aqueous solution. The structure of the ODN layer immobilized on the surface is thus of primary importance. * To whom correspondence should be addressed. Tel.: (33) 4-7272-83-64. Fax.: (33) 4-72-72-85-33. E-Mail: Hamid.Elaissari@ ens-bma.cnrs.fr. † CNRS, Lyon. ‡ CNRS, Vernaison. (1) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859. (2) Wolf, S. F.; Haines, L.; Fisch, J.; Kremsky, J. N.; Dougherty, J. P.; Jacobs, K. Nucleic Acid Res. 1987, 15, 2911. (3) Maskos, U.; Southern, E. M. Nucleic Acid Res. 1992, 20, 1679. (4) 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.

The structure of layers of adsorbed or grafted polymers on solid surfaces has been extensively studied using various experimental techniques including IR and NMR spectroscopies, surface force measurements, and radiation scattering methods.5,6 The neutron scattering techniques7 that could be applied to a large variety of polymers include small angle neutron scattering (SANS) for samples with a high specific area8-18 and the more recent neutron reflectivity measurements for macroscopic flat surfaces.19-23 The advantages of neutron scattering lie in the possibility of contrast variation and high spatial resolution as compared to experimental methods using visible light (light scattering, ellipsometry). This method appeared unique since it allows the direct measurement of the internal structure of adsorbed polymer layers and is very useful for checking the validity of different theories. In the contrast variation method, the choice of the isotopic (5) Kawaguchi, M.; Takahashi, A. Adv. Colloid Interface Sci. 1992, 37, 219. (6) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymer at Interfaces; Chapman & Hall: London, 1993. (7) Lindner, P., Zemb, T., Eds. Neutron, X-ray and Light Scattering: Introduction to an Investigative Tool for Colloidal and Polymeric Systems; North-Holland: Amsterdam, 1991. (8) Cosgrove, T.; Heath, T. G.; Ryan, K.; Crowley, T. L. Macromolecules 1987, 20, 2879. (9) Cosgrove, T.; Ryan, K. Langmuir 1990, 6, 136. (10) Auroy, P.; Auvray, L.; Le´ger, L. Phys. Rev. Lett. 1991, 6, 719. (11) Auroy, P.; Auvray, L.; Le´ger, L. Macromolecules 1991, 24, 2523. (12) Auroy, P.; Auvray, L. Macromolecules 1992, 25, 4134. (13) Auvray, L.; Cruz, M.; Auroy, P. J. Phys. II France 1992, 2, 1133. (14) Auvray, L.; Auroy, P.; Cruz, M. J. Phys. I France 1992, 2, 943. (15) Auroy, P.; Mir, Y.; Auvray, L. Phys. Rev. Lett. 1992, 69, 93. (16) Cosgrove, T.; Heath, T. G.; Ryan, K. Langmuir 1994, 10, 3500. (17) Mir, Y.; Auroy, P.; Auvray, L. Phys. Rev. Lett. 1995, 75, 2863. (18) Cosgrove, T.; Griffith, P. C.; Lloyd, P. M. Langmuir 1995, 11, 1457. (19) Guiselin, O.; Lee, L. T.; Farnoux, B.; Lapp, A. J. Chem. Phys. 1991, 95, 4632. (20) Guiselin, O. Europhys. Lett. 1992, 17, 225. (21) Field, J. B.; Toprakcioglu, C.; Ball, R. C.; Stanley, H. B.; Dai, L.; Barford, W.; Penfold, J.; Smith, G.; Hamilton, W. Macromolecules 1992, 25, 434. (22) Perahia, D.; Wiesler, D. G.; Satija, S. K.; Fetters, L. J.; Sinha, S. K.; Milner, S. T. Phys. Rev. Lett. 1994, 72, 100. (23) Karim, A.; Satija, S. K.; Douglas, J. F.; Ankner, J. F.; Fetters, L. J. Phys. Rev. Lett. 1994, 73, 3407.

10.1021/la990696d CCC: $19.00 © 2000 American Chemical Society Published on Web 12/04/1999

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Elaı¨ssari et al.

Chart 1. Structure of the (dT35) Polythymidylic Acid Bearing a Primary Amine at the 5′ Terminal Position

composition of the different components in a complex system provides a way to observe the scattering arising from one of the components in a multicomponent mixture. This can be achieved by matching the scattering length densities of all the components except that of the observed component. Thus, the adsorbed polymer layer at the surface of particles can be observed when the scattering length densities of the particles and the solvent are set identically. The second advantage of neutrons comes from their short wavelength which provides a high spatial resolution. The resolution is the size (dmin) of the smallest structural detail the experiment can resolve. It is set by the largest observed scattering vector as dmin ) π/qmax.24,25 For example, the set up used in this work gives qmax ) 1 nm-1; the resolution is thus on the order of 3 nm. The thickness of a layer thinner than 3 nm can be measured with accuracy, but any structural inhomogeneity smaller than this distance cannot be observed. In particular, the internal structure of thin layers cannot be resolved; therefore, such layers can be considered as homogeneous. The structural models used in the interpretation of the data have to account for the resolution of the experiment and should not include any structural detail smaller than the resolution. The resolution of SANS is considered as high as that of light scattering. A resolution larger than the size of the molecules (∼5 Å) would not have physical significance. In a recent paper, very interesting work has been reported by Walker et al.26 using a hydroxyl radical footprinting method to investigate the structure of singlestranded DNA block copolymer (short oligonucleotide of 40 bases with 20 uncharged nucleotides) molecules adsorbed onto polystyrene latex surfaces. The adsorption of a diblock copolymer oligonucleotide on two polystyrene latexes of opposite surface charge was studied. On the positively charged latex particles in 0.05 M NaCl solution, the ODN-block molecules were found adsorbed in a flat conformation and a large part of adsorption took place via the sugar moieties of the oligonucleotide chains. The adsorbed amount of ODN fragments depended on the architecture of the ODN, the nature of the surface charges, and the surface coverage.

In this work, small-angle neutron scattering (SANS) was used to study the conformation of adsorbed and covalently attached polythymidylic acid (dT35 bearing an aminohexyl linker at the 5′ terminus) on the latex particles bearing amino groups. The adsorption study of dT35 as a function of pH was first investigated using hydrogenated and deuterated latexes. The covalent binding was performed on deuterated cationic latex only. The structure of the layer of the adsorbed or grafted dT35 molecules was examined as a function of pH, ionic strength, and surface coverage, to highlight whether such cationic particles are suitable for biomedical diagnosis. In this paper, the adsorption isotherms, the effect of pH and ionic strength on the adsorption, and the covalent binding of dT35 onto cationic latexes are not discussed in detail, since they have already been reported and discussed in our previous papers.27-31 Experimental Section 1. Synthesis and Characterization of Polythymidylic Acid Oligonucleotides. Oligodeoxynucleotide (dT35) bearing an aliphatic primary amino group (H2N-(CH2)6-) at a 5′ terminus was synthesized on a 394 DNA/RNA synthesizer from Applied Biosystems using the standard β-cyanoethyl phosphoramidite chemistry.1 The prepared oligonucleotide was purified by reversephase HPLC using a Beckman ODS (10 × 25 mm2) column. The concentration of the purified oligonucleotides could be measured with UV absorption at 260 nm. The chemical structure of dT35 is given in Chart 1; its molecular weight is Mr ) 13416 g mol-1. 2. Preparation and Characterization of Polymer Latex Particles. The latexes used in this study were prepared by (24) Cabane, B. In Surfactant Solutions: New Methods of Investigation; Zana, R., Ed.; Surfactant Science Series 22; Marcel Dekker: New York, 1987; p 57. (25) Chevalier, Y. Trends Polym. Sci. 1996, 4, 197. (26) Walker, H. W.; Grant, S. B. Langmuir 1995, 11, 3772. (27) Elaı¨ssari, A.; Cros, P.; Pichot, C.; Laurent, V.; Mandrand, M. Colloids Surf. 1994, 83, 25. (28) Elaı¨ssari, A.; Chauvet, J.-P.; Halle, M.-A.; Decavallas, O.; Pichot, C.; Cros, P. J. Colloid Interface Sci. 1998, 202, 251. (29) Elaı¨ssari, A.; Pichot, C.; Delair, T.; Cros, P.; Ku¨rfurst, R. Langmuir 1995, 11, 1261. (30) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C. Langmuir 1997, 13, 7021. (31) Ganachaud, F. Ph.D. Thesis, Claude Bernard University, Lyon I, France, 1997.

Structure of Polythymidylic Acid on Latex Particles

Langmuir, Vol. 16, No. 3, 2000 1263

Chart 2. Oligonucleotide Coupling onto Polystyrene Latex Particle Bearing Amino Groups

emulsion polymerization of styrene using the classical emulsifierfree procedure.32-35 A deuterated polystyrene latex seed was first prepared using deuterated styrene (from Janssen Chemica) and 2,2′-azobis(2-amidinopropane) hydrochloride (V50) as the initiator. These latex particles were washed and used as seed particles in the copolymerization of deuterated styrene and vinylbenzylamine hydrochloride (VBAH) as a functional monomer using the same cationic initiator (V50). Two deuterated latexes, d-PS1 and d-PS2, and a hydrogenated polystyrene latex, (H-PS), bearing amino groups at their surface were prepared using the seed polymerization method described above. The prepared latexes were characterized with regard to particle size, polydispersity index, and surface charge density. The particle sizes were measured by transmission electron microscopy at the CMABO (University of Lyon I) using Hitachi TEM equipment. The surface charge densities were determined by the N-succinimidile ester 3-(2-pyridyldithiol) propionic acid (SPDP) colorimetric titration method.34,35 The details on the synthesis, the copolymerization process, and the characterization of H-PS have been reported elsewhere.32,33,35 3. Adsorption Measurements. The adsorption of dT35 onto latex particles was measured using the depletion adsorption method. The adsorbed amounts NS (mg m-2) were calculated from eq 1

NS )

V(Ci - Cf) MASP

(1)

where V (mL) was the volume of the solution, Ci (mg mL-1) and Cf (mg mL-1) were the initial and the final concentrations of dT35 in the solution, respectively, ASP (m2 g-1) was the specific surface area of the latex particles and M (g) was the mass of latex in the suspension. The adsorption was studied in a phosphate buffer at a given pH and ionic strength. The adsorption measurements were performed as follows: after 3 h of incubation at 25 °C, the latex particles and the supernatant were separated by centrifugation and the concentration of free oligonucleotides in the supernatant was determined by optical density measurements at 260 nm using an HPLC system (Gen Pak Fax column from Waters) equipped with a UV detector (Uvikon 930 spectrophotometer). 4. Covalent Binding Measurements. The chemical binding of oligonucleotides onto the latex particles bearing amino groups was performed as follows: the terminal primary amino groups of the dT35 molecules (bearing an amino-linker at position 5′) were activated using 2,4-phenylene diisothiocyanate (DITC) as (32) Ganachaud, F.; Sauzedde, F.; Elaı¨ssari, A.; Pichot, C. J. Appl. Polym. Sci. 1997, 65, 2315 (33) Sauzedde, F.; Ganachaud, F.; Elaı¨ssari, A.; Pichot, C. J. Appl. Polym. Sci. 1997, 65, 2331 (34) Delair, T.; Marguet, V.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1994, 272, 962. (35) Ganachaud, F.; Mouterde, G.; Delair, T.; Elaı¨ssari, A.; Pichot, C. Polym. Adv. Technol. 1995, 6, 480.

an activating agent before adding the suspension of latex particles having amino groups at their surface.31,36 The particles used were either bare or post-stabilized by adsorption of a nonionic emulsifier Triton X-405 (d-PS2 series). The principle of the covalent binding is given in Chart 2. The immobilized amount of dT35 on the latex surface was determined by measuring the difference between the initial and the final concentration of the dT35 in the supernatant in the same way as in the adsorption measurements. The latex particles studied by SANS were cleaned by repetitive centrifugations. 5. Small Angle Neutron Scattering. 5.1. Experimental Setup. The SANS experiments were carried out with the PACE spectrometer at the Laboratoire Le´on Brillouin (LLB) in Saclay.37 The samples contained in Hellma quartz cuvettes were illuminated with a neutron beam of 0.7 cm in diameter defined by the collimation apertures, and the scattered neutrons were collected on a detector consisting of 30 concentric rings, 1 cm each width. The observed scattering vector (q ) (4π/λ)sin(θ/2)) domain was defined by the wavelength λ ) 6.48 Å and the sampleto-detector distance SD ) 3 m (collimation distance ) 2.5 m), giving the experimental q-range as 0.1 nm-1 < q < 1 nm-1. The wavelength dispersion ∆λ/λ of the velocity selector was 10%. The collected data were normalized for the detector efficiency using the isotropic scattering of a 1 mm thick calibration sample of H2O. This also provides a first estimate of the scattered intensity on an absolute scale according to Jacrot et al.38 Thus, the scattered intensity was normalized to that of the H2O sample and multiplied by the value of the scattered intensity of H2O calculated from the transmission of the sample. The transmission of an H2O sample of thickness d ) 1 mm was T ) 0.53, giving IH2O ) (1/4πd)((1 T)/T) ) 0.70 cm-1. These data reductions were performed with standard procedures available as program packages at the LLB, giving the differential scattering cross-section dσ/dΩ ) I(q) after subtraction of the incoherent background.39 Because the incoherent scattered intensity is not perfectly isotropic, the exact scattered intensity of H2O in the small angle domain has to be corrected by a factor (f) which depends on the experimental setup and the wavelength and is generally found from 0.5 to 1.38 This factor will be estimated from the scattering of a PS latex suspension of given diameter under known contrast conditions used as a calibration sample, as described in the following. 5.2. Contrast Conditions. The scattering length densities of the deuterated polystyrene particles and the dT35 oligonucleotide have to be estimated. In particular, the accuracy of the contrast matching of the PS particles by the solvent was carefully checked (36) Delair, T.; Meunier, F.; Elaı¨ssari, A.; Charles, M. H.; Pichot, C. Colloids Surf. 1999, 153, 341. (37) E Ä quipements Expe´ rimentaux du Laboratoire Le´ on Brillouin; available from LLB, CEN Saclay, 91191 Gif-sur-Yvette Cedex, France. The LLB is a CEA-CNRS laboratory. (38) Jacrot, B.; Zaccai, G. Biopolymers 1981, 20, 2413 (39) Cotton, J.-P. In Neutron, X-ray and Light Scattering: Introduction to an Investigative Tool for Colloidal and Polymeric Systems; Lindner, P., Zemb, T., Eds.; North-Holland: Amsterdam, 1991; p 19.

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Elaı¨ssari et al. Table 1. Scattering Length Densities used in This Work compound

scattering length density (cm-2)

protonated water 1H2O deuterated water 2H2O deuterated polystyrene H2N(CH2)6-dT35 C6DTU-dT35

- 0.559 × 1010 6.384 × 1010 6.507 × 1010 2.917 × 1010 2.935 × 1010

length density of deuterated water. It is thus not possible to achieve the complete matching of the PS particles by using H2OD2O mixtures; the zero-contrast condition corresponds to a hypothetical mole fraction of D2O of 102% (as calculated from eq 3). As a consequence, the experimental data will include a scattering coming from the PS particles, even when the solvent is pure D2O. This scattering decays as q-4 (Porod’s law) and is not negligible in the present cases where the scattering coming from the adsorbed polymer layer is weak. The scattering length density of dT35 was calculated from the scattering lengths of nuclei and the partial molar volumes of the monomer unit of polythymidylic acid and of the linker. The volume of the dT monomer unit is taken as the sum of the experimental partial molar volumes of thymidine (167.90 cm3/mol)41 and hydrogenophosphate monoanion H2PO4- (29.1 cm3/mol).42 For the adsorption experiments, the oligonucleotide bears an aminohexyl linker C6NH2,

located at the 5′ terminus; while the bridging group for the grafted oligonucleotide is a hexylphenylenedithiourea moiety C6DTU.

Figure 1. a: Scattered intensity of 1% suspensions of the d-PS1 particles in water of the following different isotopic compositions xD2O (O: 70%; +: 80%; b: 90%; *: 95%; ]: 100%;). The scattered intensity is linear in the log-log plot with a slope of -4 according to Porod’s law. b: xAPrd as a function of FS allowing the determination of FPS ) 6.507 × 1010 cm-2.

with FD2O ) 6.384 × 1010 cm-2, FH2O ) -0.559 × 1010 cm-2. The conventional contrast variation method makes use of the zero angle scattering I(0) which requires the extrapolation of the experimental data to q ) 0 according to Guinier’s law. In the present case where Porod’s law is complied with in the full q-range, such an extrapolation cannot be made and the prefactor of Porod’s law (APrd) was used instead of I(0). The contrast matching conditions where FS ) FPS were determined in a plot of xAPrd as a function of FS (Figure 1b). FPS ) 6.507 × 1010 cm-2 was found, in agreement with the value FPS ) 6.476 × 1010 cm-2 which can be calculated from the scattering lengths of carbon (0.6648 × 10-12 cm) and deuterium (0.6674 × 10-12 cm) and the density of polystyrene-d8 (1.13 g/cm3). The important point is that this value exceeds the scattering

The volumes of these two linkers, as calculated from tables of group contributions to the partial molar volume,43 were taken as 104.7 and 238 cm3/mol, respectively. Since all the experiments were carried out in D2O, the labile OH and NH protons of the oligonucleotides were deuterated by isotopic exchange with the solvent. The calculation of the scattering length densities takes the isotopic exchange into account and is thus only valid for samples in D2O. The scattering length densities are summarized in Table 1. For the adsorption experiments, the labile protons of the H2N(CH2)6-dT35 oligonucleotides were exchanged against D2O by repeated dissolutions and dryings. The preparation of the samples consists of the simple mixing of a solution of H2N(CH2)6-dT35 in D2O with a suspension of latex in D2O. Since both isotopic compositions could be set close to that of the supplier’s quality (D2O of isotopic purity >99.9% from Spectrome´trie Spin et Techniques), the contrasts are as given in Table 1. The situation is different for the grafting experiments since the grafting reaction was carried out in 1H2O and the deuteration of the solvent was done by repeated cycles in which the suspension was diluted in D2O and concentrated by evaporation under vacuum. The isotopic composition of the solvent was thus different for each sample and was measured by 1H NMR. Thus, the integrations of the 1H NMR lines of HDO (at 4.5 ppm/TMS) and of dioxane (at 3.7 ppm/ TMS) added in a known amount as an internal integration reference, give the concentration of residual protons. The scattering length density of the solvent was then calculated from the isotopic composition by eq 3. 5.3. Absolute Intensity Scaling. Since the radius R of the polystyrene particles was measured by means of electron microscopy and the volume fraction of particles is known (φPS ) 1), the contrast variation experiment used for the determination of FPS allows a correct scaling of the scattered intensities on an absolute intensity scale. Indeed, the incoherent scattering of H2O

(40) Williams, C. E. In Neutron, X-ray and Light Scattering: Introduction to an Investigative Tool for Colloidal and Polymeric Systems; Lindner, P., Zemb, T., Eds.; North-Holland: Amsterdam, 1991; p 101.

(41) Patel, S. G.; Kishore, N. J. Solution Chem. 1995, 24, 25. (42) Millero, F. J. Chem. Rev. 1971, 71, 147. (43) Cabani, S.; Gianni, P.; Mollica, V.; Lepori, L. J. Solution Chem. 1981, 10, 563.

by means of the contrast variation method.40 The scattering of the d-PS1 particles with a 220 nm diameter follows the asymptotic Porod’s law in the observed q-range (Figure 1a)

A I(q) ) 2π(FPS - FS)2 q-4 ) APrdq-4 V

(2)

where A/V is the interfacial area per unit volume of sample. The variation of the scattering length density of the solvent (FS) was obtained by means of H2O-D2O mixtures of different compositions where

FS ) xD2O FD2O + (1 - xD2O)FH2O

(3)

Structure of Polythymidylic Acid on Latex Particles

Langmuir, Vol. 16, No. 3, 2000 1265

used for the intensity calibration has to be corrected by a factor f because it is not perfectly isotropic. Porod’s law (eq 2) can be rewritten as follows due to the spherical shape of the PS particles

I(q) ) 2π(FPS - FS)2

3φPS -4 q ) APrd q-4 R

(4)

The experimental scattered intensity obtained, assuming that f ) 1, differs from the calculated values by a factor f. This allows a precise determination of f from the slope dxAPrd/dFS, giving f ) 0.76. The corrected incoherent scattering of H2O is then IH2O ) 0.70/0.76 ) 0.92 cm-1, close to the value of 0.87 cm-1 reported in the literature.44 The slight difference between these two values may be ascribed to the weak but systematic underestimation of radii by electron microscopy. All the experimental data presented in the following have been corrected by this factor. 5.4. Theory of Small Angle Neutron Scattering. The theory of the small angle neutron scattering as applied to adsorbed or grafted polymer layers is widely described and discussed in the literature.8,11,45 A short theoretical background with the equations used in the present work is given in this section. The total scattered intensity I(q) of a suspension of particles coated with an oligonucleotide layer (by either adsorption or grafting), can be split into three partial structure factors related to the particle-particle, particle-ODN, and ODN-ODN correlations

I(q) ) IP-P(q) + IP-O(q) + IO-O(q)

(5)

The momentum transfer is related to the scattering angle θ and the wavelength λ by

q)

θ 4π sin λ 2

()

(6)

In the wide q-range of the present work, the particle-particle structure factor IP-P(q) complies with Porod’s law

3φP -4 A q (7) IP-P(q) ) 2π(FP - FS)2 q-4 ) 2π(FP - FS)2 V R where FP and FS are the scattering length densities of the particles and the solvent, respectively. The interfacial area per unit volume of sample, A/V, is related to the radius R of the spherical particles and the volume fraction of particles φP by simple geometry. The scattered intensities IO-O(q) and IP-O(q) for the ODNODN and ODN-particle interferences over all orientations can be expressed as a function of volume fraction profile of the oligonucleotides fixed onto the surface

IO-O(q) ) 2π(FO - FS)2

3φP -2 q | R

IP-O(q) ) -4π(FP - FS)(FO - FS)

∫

∞

0

φ(z)eiqz d z|2 + I˜O-O(q) (8)

3φP -3 q R

∫

∞

0

φ(z) sin(qz) d z (9)

where FO is the scattering length density of the ODN and φ(z) is the average ODN volume fraction perpendicular to the particle surface (in the z direction). The correlation structure factor I˜O-O(q) which comes from the polymer concentration fluctuations inside the adsorbed layer is most often small but contributes significantly to the scattering when qH .1 (where H is the layer thickness).46 This regime cannot be reached when the layer is very thin and this term was neglected in the following. The particle-particle and ODN-particle partial structure factors vanish at the match point where FP ) FS. But this condition (44) Ragnetti, M.; Geiser, D.; Ho¨cker, H.; Oberthu¨r, R. C. Makromol. Chem. 1985, 186, 1701. (45) Auvray, L.; Auroy, P. In Neutron, X-ray and Light Scattering: Introduction to an Investigative Tool for Colloidal and Polymeric Systems; Lindner, P., Zemb, T., Eds; North-Holland: Amsterdam, 1991; p 199. (46) Auroy, P.; Auvray, L. J. Phys. II France 1993, 3, 227.

Table 2. Characteristics of the Latex Particles: Surface Charge Density, Particle Size and Uniformity Ratio latex

σ (µmol cm-2)a

DN (nm)

DW (nm)

Ub

H-PS d-PS1 d-PS2

1.76 1.55 1.04

241 220 142

243 221 144

1.005 1.002 1.005

a Surface density of amine and amidine groups. b Uniformity ratio.

could not be fulfilled in the present case, so that the full three terms were taken into account. In the case where the volume fraction of ODN in the adsorbed layer is assumed to be constant, the ODN volume fraction profile is a step function defined by φ(z) ) Ns/H for z < H and φ(z) ) 0 for z > H, the IO-O(q) and IP-O(q) intensities read

IO-O(q) ) 4π(FO - FS)2

( )

3φP -4 Ns q R H

IP-O(q) ) -4π(FP - FS)(FO - FS)

2

[1 - cos(qH)]

(10)

3φP -4 Ns q [1 - cos(qH)] R H (11)

In this condition, the structure of the adsorbed layer is fully described by the only two remaining parameters, the adsorbed (or grafted) amount of oligonucleotide (NS) and the mean thickness of the layer (H). The amount of interfacial ODNs (NS) and the thickness (H) of the layer can be determined from the total scattered intensity I(q) as a function of scattering vector q by taking into account the scattering length densities of the ODNs, colloidal particles, and the contrast matching conditions set by the solvent isotopic composition. At low q-values where the condition that qH , 1 is fulfilled, the expressions of q2 IO-O(q) and q2 IP-O(q) derived from eqs 10 and 11 are both second degree polynomials

q2 IO-O(q) ) 2π(FO - FS)2

[

]

3φP 2 q2H2 Ns 1 R 12

q2 IP-O(q) ) -2π(FP - FS)(FO - FS)

[

(12)

]

3φP q2H2 Ns H 1 R 12

(13)

Results and Discussion The adsorption mechanism and the covalent binding studies of dT35 molecules onto polystyrene cationic latex particles have already been presented in previous papers.27-31,47 The adsorption study of dT35 was carried out using hydrogenated and deuterated latexes in order to check against any isotopic effect of deuterated polystyrene on the adsorption. Covalent binding was performed on deuterated latex only. To investigate the conformation of adsorbed and grafted oligonucleotides, the SANS study was performed on the deuterated polystyrene latexes using D2O as a contrast matching agent. 1. Characteristics of the Latex Particles. The size and electrical charge of the latexes after a cleaning step are reported in Table 2. The particles are monodisperse in size as reflected by the uniformity ratio close to 1. According to the polymerization method (seed polymerization) used for the preparation of these latexes, the cationic groups originated mostly from the vinylbenzylamine hydrochloride rather than from the cationic initiator. 2. The Effect of pH on the Adsorption of ODN onto the Latex Particles. The adsorption of oligonucleotide (dT35) onto H-PS and d-PS1 latexes was examined as a (47) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C.; Laayoun, A.; Cros, P. Langmuir 1997, 13, 701.

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Langmuir, Vol. 16, No. 3, 2000

Figure 2. Maximal adsorbed amount (Ns) of dT35 onto (b) H-PS and (0)d-PS1 as a function of pH (10-2 M ionic strength, 25 °C). Residual adsorbed amount (Ns) of dT35 on the (9) d-PS2 a function of pH, after two washes using the adsorption buffer.

function of pH at a constant ionic strength (NaCl 0.01 M). The deuteration of the polystyrene does not influence significantly the adsorption of dT35, as shown in Figure 2, in which the maximal adsorbed amounts of dT35 (at the plateau of the adsorption isotherms) as a function of pH are reported. The adsorption is higher in an acidic medium than in an alkaline one. In the investigated pH range (from pH 3.5 to 9.0) the ODN are negatively charged, whereas the charge density of the cationic particles depends on the pH. At low pH (pH < 7) where the protonation of the amino groups on the latex surface is complete, the adsorbed amount of dT35 is high. The adsorbed amount of dT35 drastically decreases as the pH increases, since the latex surface becomes less charged. This result corroborates that electrostatic interactions are the driving forces in the adsorption process. The contribution of hydrophobic interactions to the adsorption is not negligible since adsorption is still quite high in basic media as already reported in refs 27-30 and by Walker et al.26 On this basis, the slight differences in the adsorption of dT35 on the d-PS1 and H-PS latex particles (Figure 2) can be attributed to the slight difference in their surface charge densities (1.7 and 1.5 µmol cm-2 for H-PS and d-PS1 respectively). In addition, the adsorption of dT35 was shown to be quite strong (irreversible) whatever the pH since the residual adsorbed amounts of dT35 onto latex particles remained only slightly lower than the initial adsorbed quantities even after two washes using the adsorption buffer for a given pH (Figure 2). 3. Conformation of the Adsorbed and Grafted ODN Layers. Small-angle neutron scattering was used to determine the structure of the adsorbed or grafted dT35 layer on the latex surface. All SANS measurements were performed using d-PS1 and d-PS2 latexes in a deuterium oxide (D2O) medium. The effect of the pH, ionic strength, and the immobilized amount of dT35 were investigated. Sensitivity of the SANS Method As mentioned in the theoretical section, the spatial resolution of the experiment is in the region of 30 Å according to the investigated q-range. The resolution is thus rather low compared to the dimensions of the oligonucleotide (the length of the fully extended ODN is around 120 Å in helical conformation). As a consequence, the experiment is not sensitive to the internal structure of the interfacial (adsorbed or grafted) layer and only two structural parameters can be determined from the data: the immobilized amount NS and the mean thickness of the interfacial layer H. The interfacial layer is thus

Elaı¨ssari et al.

Figure 3. Simulations of the effect of the layer thickness (H) and incoherent background (B) subtraction on the scattered intensity (plot of q2I(q) vs q2).

considered as homogeneous according to the experimental resolution. The scattered intensities IO-O(q) and IP-O(q) are described by eqs 10 and 11 where both parameters NS and H appear clearly. The adsorbed (or grafted) amount NS is obtained with good accuracy by an extrapolation of q2[IP-O(q) + IO-O(q)] to q ) 0. The uncertainty on the NS values comes from errors in the SANS measurement itself, in the absolute intensity scaling and in the determination of the scattering length density of the ODN. In contrast, the accuracy of the thickness determination and even the possibility of measuring the thickness of layers thinner that the resolution is raised. It is worth discussing this point prior to giving the results. Thus, in the cases where H is small, IO-O(q), which is the predominant term according to the present contrast conditions, reduces to eq 12 of the form IO-O(q) ) a[(1/q2) - (H2/12)] where a is a constant independent of q and H (the domain of validity of eqs 12 and 13 extends over the whole experimental q-range). H only appears in the second term which does not depend on q. The effect of a variation of H is thus to shift the scattered intensity by a constant, in the same way as in a background subtraction. Thus, one cannot distinguish between a variation of H and a slight error in the estimation of the incoherent background B. If the background B is not precisely known and is used as an adjustable parameter, the slope of q2 IO-O(q) vs q2 (eq 12) gives B - (H2/12). In brief, the thickness H can be determined accurately when it is large enough for significant deviations from eq 12 to occur or when the contrast is such that there is a significant contribution of the crossed term IP-O(q). The results of simulations using eqs 10 and 11 (Figure 3) show that the scattering from a layer of 20 Å thickness cannot be distinguished from the scattering from a layer of 30 Å thickness, to which a background of 0.15 cm-1 has been added. On the contrary, significant differences between a layer of 40 Å thickness and one of 50 Å with a background of 0.2 cm-1 appear for q > 0.07 Å-1 because of the curvature of q2 × I(q) vs q2. The minimum thickness which can be measured is estimated as 40 Å. First, the comparison of the bare latex particles and particles coated by adsorbed oligonucleotide molecules (Figure 4) allows us to check against the sensitivity of the SANS measurements. As shown in Figure 4 in log scale, the scattered intensity I(q) is clearly higher for latex particles bearing dT35 molecules than for bare latex. The presence of interfacial dT35 molecules on the latex particles is the origin of the scattered intensity, whereas the scattered intensity from the bare latex was principally attributed to the presence of a small amount of hydrogenated functional monomer (VBAH) on the latex surface.

Structure of Polythymidylic Acid on Latex Particles

Langmuir, Vol. 16, No. 3, 2000 1267 Table 4. Effect of the Grafted Amount of dT35 onto Latex Particles, pH, and Ionic Strength on the Thickness Layer Effect of the Grafted Amount of dT35 onto d-PS1 on the Thickness Layer at pH 9.2 and 0.01 M NaCl sample

% D2O

Ns (mg m-2)

HT (Å)

0C 1C 2C 3C

97.04 99.00 96.90 94.05

0.00 0.34 0.69 0.87

s 40-50 50-60 40-80

Effect of pH on the Thickness Layer at pH 9.2, 0.01 M NaCl and for a Constant Grafted Amount of dT35 onto d-PS2

Figure 4. Scattered intensity (I(q) vs q in log-log scale) of the bare and coated (d-PS2) latex particles.

sample

pHa

% D2O

Ns (mg m-2)

HT (Å)

1C 3C 5C

5 7 9

98.90 99.00 99.82

0.29 0.35 0.29

40-50 40-50 40-50

Table 3. Effect of Adsorbed Amount and pH on the Thickness Layer of dT35a effect of adsorbed amount of dT35 onto d-PS1 at pH 6.0 sample

%D2O

Ns (mg m-2)

15A 16A 17A 18A 19A

99.60 99.66 99.68 99.59 99.55

0.00 0.04 0.20 0.40 0.80

HT (Å)