Results and Discussion

The volume of solution analyzed by the DLS system (crossing space of laser and detector) has been estimated to be around 1 pL [88], which is equivalent to a sphere of approximately 12.5 micrometers in diameter. This volume is not negligible for sub-millimeter diameter capillaries, especially in the case of 0.1 mm capillaries that, on the other hand, are the most widely used because of its small sample consumption (approximately 400 nL). It is therefore required to ensure that no spatial distortions occur during measurement to obtain a clean DLS signal, similar to that obtained in quartz cuvettes, considering the geometry of the capillary and the plastic walls of the GCB-D. Capillaries of 0.1, 0.2 and of 0.3 mm, with and without agarose,

23 were filled with the supernatant of a centrifuged protein solution of 50 mg/mL in its buffer. The concentration of the supernatant was measured to be of 49 mg/mL (Absorption at 280 nm). After this, the different capillaries were punctured in GCB-D’s filled with 3 M ammonium sulfate. DLS signals were acquired at 30 mm from the open end of the capillary (Figure 9). To make sure that the precipitant concentration in the observed volume was negligible, the DLS measurements were performed 5-15 minutes after puncturing the GCB-D. An aliquot of the protein sample in its buffer without precipitant was also measured by DLS in a quartz cuvette for comparative purposes. For all the capillaries, DLS signals were measured for 30 seconds, 30 consecutive times and 2 seconds interval between measurements. The results of the size distributions obtained from the DLS signals are shown in Figure 9. Figure 9C shows how the focus of the DLS laser beam fits inside the capillary, being sufficiently away from the flares caused by the reflection of the laser on the walls of the GCB-D, even in the case of the smallest 0.1 mm capillaries. Figure 9A shows an overlay plot of the radius distribution of glucose isomerase in capillaries and the optical cuvette.

Figure 9A indicates that the size distributions calculated from DLS measurements inside the cuvette and those taken inside the capillaries are comparable. In all cases a monomodal size distribution was observed in which the only peak evident is that corresponding to the hydrodynamic radius of glucose isomerase in the buffer without precipitant.

Figure 9: Results of the DLS measurements in capillaries. A). Comparison of the size distributions obtained from DLS measurements of the same protein solution in standard quartz cuvettes and inside capillaries. The measured radius was approximately 2 nm in all 5 cases. B) ACF of the respective measurements, C) shows the focus of the laser inside a 0.1 mm capillary: the white dot marks the focus of the collecting optics and is slightly off for the sake of clarity, of 0.1 mm 0.2 mm, 0.3mm and 0.3mm with agarose.

Figure 9B shows an overlay of the auto correlation functions from which the radius distributions were derived. One can see that the signal to noise ratio for DLS

24 measurements in the cuvette is a bit better than for measurements in capillaries but all the ACF are shaped as expected [54, 56, 91] for monomodal solutions. These results clearly demonstrate that DLS measurements can be performed inside capillaries with an inner diameter as small as 100 µm. Moreover it could be shown that it is possible to measure DLS in standard commercial counter-diffusion devices.

After proving that DLS measurements can be performed inside capillaries a counter-diffusion crystallization experiment was set-up and the evolution of size distribution of the protein as a function of time and distance inside the capillary was monitored by DLS. A 0.1 mm capillary was filled with glucose isomerase solution at a concentration of 50 mg/mL in buffer, then punctured it in a GCB-D box containing 3 mol/L ammonium sulfate as precipitant and proceeded as described in the experimental section. The results of this experiment can be seen in Figure 10.

As soon as the open-end capillary is put in contact with the precipitant solution, the protein and the mixture start to counter-diffuse against each other. The precipitant diffuses faster than the protein due to its larger diffusion coefficient (approximately one order of magnitude). As the precipitant travels through the capillary, the solubility of the protein decreases and precipitation takes place. The calculated size distributions seem to react to this process in two different ways. At first, the increasing concentration of the precipitant causes an increase in the hydrodynamic radius of the protein (from 2 nm to approximately 5 nm).

A possible explanation for this observation is that the higher number of ions in the solution, their interaction with the protein and the protein-protein interactions mediated through the electrostatic conditions of the solution influence the measured diffusion coefficient, which is used by SPECTRO to calculate the hydrodynamic radius RH using the Stokes-Einstein Equation [91]. The difference of theoretical RH

and measured RH is, at constant protein concentration, a function of precipitant concentration (see chapter 1.7). Thus by relating measured RH with theoretical RH of the Protein it is in principle possible to determine the concentration of precipitant [61].

The second effect is that, at a certain time during the counter-diffusion, there is a perturbation of the size distribution consisting in the prompt extinction of the peak corresponding to the protein in solution and in the appearance of broad peaks at larger diameters.

25

Figure 10: Size distribution (X-axis) as a function of time (Y-axis) obtained from DLS measurements in a single capillary. The number at the bottom right corner of each picture indicates the distance of the measurement in mm from the open end of the capillary. The picture at the bottom shows an overview of the capillary and the position of the measurements (*) in the GCB-D.

The fast disappearance of the initial protein peak is indicative of crystallization events [88] that seem to take place progressively later in time at larger distances from the entrance of the capillary, as expected from the experimental observation and the simulation results. The irregular size distributions at larger hydrodynamic radii correspond to the perturbation of the DLS signal by the appearing crystals. This is consistent with the expected formation and evolution of the already described advancing supersaturation wave [98] produced by the continuous diffusion of the precipitant. At the largest distances investigated it can be seen that the crystallization is not so clearly observed from DLS data. The reason for this may be that the process of nucleation and crystal growth at those positions occur at lower super saturation conditions than for points closer to the entrance. Since those processes are the cause of the depletion of the protein, it can be concluded that, at longer distances within the capillary, they are not fast and/or intense enough to counteract the restoration of the consumed protein by diffusion from the rest of the capillary.

26 Nevertheless, the rise in concentration of the precipitant due to diffusion can be clearly observed in the increase of RH.