Small Angle Neutron Scattering Analysis

The conformation of the immobilized ODN molecules (adsorbed or grafted) on the deuterated polystyrene latexes was analyzed using SANS on three types of sample: bare latex, latex bearing adsorbed ODN, and latex carrying chemically grafted ODN [19].

Since the neutron scattering of the latex particles was not completely nil and the quantities of ODN adsorbed or grafted were very low, each measurement required considerable counting time. However, a systematic approach during data processing combined with knowledge of given variables (length of scattering of certain species, quantities of ODN immobilized) made it possible to draw out a trend on ODN surface conformation.

The latexes carrying the adsorbed or grafted ODN were then analyzed at acidic pH (i.e., pH 5), where the scattered intensity I(q) is shown as a function of scattering vector (q) in Fig. 24. The curves in the figure are obtained from a model in which the thicknesses of the ODN layer are 20, 50, 80, and 120 A, respectively. The results show that under these conditions the conformation of adsorbed ODN is neither "brush-like" (which would be the case for h = 150 A) nor completely flat (for h = 10 A). A mean thickness from 50 A to 70 A correlated with the experimental results depending on the ODN-immobilized amount. Varying the medium's ionic strength and pH had no effect on adsorbed or grafted ODN layer thickness.

The conformation of ODN adsorbed onto anionic and cationic polystyrene latexes particles was first reported by Walker et al. [20,21]. Preliminary determination of the ODN layer thickness on latex surface by photon correlation spec-

FIG. 24 Scattering intensity I(q) vs. q in log-log scale of deuterated polystyrene particles where dT35 was (a) adsorbed at pH 5 and 0.01 M NaCl, and (b) covalently grafted at pH 9.2 and 10-2 M NaCl. (From Ref. 19.)

troscopy failed, due to the poor sensitivity of the method and using such small polyelectrolytes. A second method issued from biology, i.e., hydroxyl radical footprinting, was adequately used. It consists of cleaving ODNs by a radical source and analyzes the length and distribution of the obtained fragments by gel-blot migration. In the present case, cleaving did not occur for ODN segments wholly adsorbed on the surface, which is a way to differentiate trains from loops and tails. The conformation of ODN adsorbed on the surface of cationic or anionic polystyrene latexes shown to be flat in both cases. ODN can be adsorbed either by its charges (electrostatic forces) or by its nucleic bases (stacking effect between the aromatic compounds of the bases and styrene), so that ODN and the colloidal support are in close contact.

Recently, Charreyre et al. [22] investigated the conformation of ODN chemically grafted on an amino-containing polystyrene latex using fluorescence energy transfer. A couple of fluorescent molecules, fluorescein (donor) and tetramethylrhodamine (acceptor), are grafted to the 3' end of the ODN and on amino groups on the latex surface, respectively. The energy transfer between acceptor and donor varies with the mean distance between the two fluorophores, i.e., between the ODN and the surface, so that a semiquantitative study is envisaged. Prior to grafting reactions, latexes were stabilized by nonionic surfactant (Triton X-405) which, even after extensive centrifugation/redispersion steps, was not fully eliminated (see discussion on desorption). In these conditions, a "brush"-type structure was observed at a basic pH (pH 10), whereas at neutral or weak acid pH (pH 6) conformation is mostly flat.

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