It has been demonstrated that CNTs are able to interact with surrounding water molecules275,276 and to influence the relaxation time of the latter, depending on the functional groups exposed on their surface.277,278 Therefore the T2 spin-spin NMR relaxometry of protons in water, being sensitive to the constraints of the molecule,279 can be a useful technique to quantitatively evaluate concentration, dispersion and aggregation of f-CNTs in water solutions. In fact, the stability of f-CNTs in water strongly depends on the nature and extent of their chemical substitution, in particular with polar and even ionic groups to reduce the unfavorable solute-solvent interactions.
Visual inspection of suspensions of the f-CNTs in water at the concentration of 0.1 % (w/V) reveals such tendency, in particular for the pristine, un-derivatized f0-CNTs. Moreover, aggregation manifests as a significant time-dependent effect; sonication helps producing a visible dispersion of f-CNTs.
RESULTS AND DISCUSSION
The effect of CNTs on the relaxation time of water and the aggregation tendency in aqueous systems has been evaluated measuring T of the dispersions at different time intervals. f-CNTs 2
have been dispersed in pure water and in culture medium (complete DMEM in water), in view of future studies on CNTs in cell-containing systems. Moreover, the chosen f-CNTs have been dispersed in an aqueous alginate solution (2% w/V) in order to evaluate the ability of LF-NMR to detect the aggregation tendency of CNTs in this condition. The reasons for choosing alginate are manifold: i) alginate is a very hydrophilic polymer, and as such it may severely affect the structuring of water and the ensuing effect on the solubility of co-solutes; ii) alginate is an anionic polyelectrolyte, and its negative charges may trigger different interactions with differently charged CNTs; iii) alginate is one of the most important biopolymers used in the preparation of scaffolds and, in general, in tissue engineering, which may be interesting areas of application of CNTs.
In general, T was found to be notably lowered upon addition of CNTs although such effect 2
strongly depended on their dispersion state (the T values recorded for water and for f-CNTs-free 2
aqueous solutions are quite long: water, ~ 2400 ms; DMEM, ~ 2200 ms; alginate, ~ 1400 ms).
Preliminary experiments were carried out for the various f-CNTs, in the absence or in the presence of co-solutes (DMEM or alginate), adjusting the sonication time for each case to the goal of reaching a visual disappearance of massive aggregation (i.e. good dispersion). The results are reported in Figure 15, A to C.
RESULTS AND DISCUSSION
Figure 15. Time dependence of the average relaxation time (T2) of dispersions of f0-CNTs (A), f1-CNTs (B) and f2-CNTs (C), in pure water (), in DMEM (▲) and in alginate ().
f-CNTs concentration: 0.1 % (w/v).
As general statements, i) the T values of all f-CNTs show a more or less marked time-2
dependence; ii) the range of T values span over two orders of magnitude, from about 45 ms (for 2
f1-CNTs in DMEM) to that of pure water (for f0-CNTs at long times). Correspondingly, it is possible to identify three extreme types of behavior. In pure water, f0-CNTs display T values, 2
which started from values close to that of pure water, for further increase during the time interval of the experiment, practically reaching the value of the relaxation time of free water molecules after 40 minutes. The latter effect seemed to be clearly correlated with the ongoing massive macroscopic aggregation of CNTs.
RESULTS AND DISCUSSION
f0-CNTs in DMEM, and both f1-CNTs and f2-CNTs in all conditions: i) show much lower T 2
values than f0-CNTs in water, indicating an initial condition of much better dispersion. As demonstrated by Cheng et al.,280 serum proteins are able to adsorb on the CNTs surface, a phenomenon that leads to a better dispersion of the CNTs;281 ii) seem to conform to an exponential increase of T2 values with time (this is evident by plotting the data as a linear function of time:
data not shown). This behavior can be clearly associated with an increasing association, however starting from values much lower than those of un-derivatized f0-CNTs in pure water. A discussion of the specific effect of the co-solute (DMEM or alginate) on the T2 values is postponed to the following paragraph.
Finally, the time dependence of the T2 values of f0-CNTs in aqueous alginate show a peculiar behavior, inasmuch as after some sort of induction time of about 40 min, they show an abrupt increase which sigmoidally reaches a plateau at T2 values not very far from those of water in aqueous alginate at 2% concentration. This behavior is at variance with both above described case:
it is tempting to suggest an all-or-none equilibrium between two states of solvation/interaction with alginate. In an initial step, f0-CNTs might be rather well dispersed, until the rapid onset of a reorganization of the CNTs around the polyanion (which very rapidly achieves its final
“equilibrium” condition), characterized by an overall effect on the relaxation time of water very similar to alginate itself. It should be recalled that the mass ratio of alginate-to-CNTs is 20:1, thus making the dominant role of the ionic polysaccharide very plausible. Given the limited time window of the experiments, it is not possible to make any speculation if also in the cases of f1 -CNTs and f2-CNTs with alginate, the observed strong increase of T2 values at long times would also lead to some sort of plateau close to the T2 value of CNT-free alginate solution, with generality of the process.
Overall, comparing the relaxation results with the visualization of the f-CNTs dispersions over time, it can be concluded that T2-NMR is a reliable technique to detect aggregate formation.
RESULTS AND DISCUSSION
3.1.2 CORRELATION BETWEEN r2 AND CONCENTRATION OF FUNCTIONALIZED