Representative results obtained using an Imidazole buffer are presented in . Panel a shows the results obtained with a negatively charged cation exchange bead, while panel b was obtained using a positively charged anion exchange bead. In both situations, negatively charged sulfate microspheres were used.
Figure 1 Representative optical images of colloidal particle exclusion zones formed around a) negative and b) positive ion-exchange beads. Images captured 90 seconds after introducing the bead into the microsphere suspension (pH 7.0, 10 mM Imidazole, 0.5 μm (more ...)
Substantial EZs developed over several minutes in both cases (). The EZs formed in the shape of an enveloping shell. Their thickness was approximately 200 μm. Microspheres were fully excluded from these zones and only rarely was the occasional particle trapped within. The concentrated ring of particles at the boundary () eventually diffused away after 10 – 15 minutes, leaving the exclusion zone clear and the surrounding suspension filled with a uniform distribution of microspheres. At low buffer concentration the boundary ring was less well defined and constituent microspheres typically diffused away more quickly than at higher buffer concentrations.
Dynamics of EZ formation are plotted in . Initially, the rate of formation was very high (10 μm/sec); then, it slowed considerably, the EZ reaching a plateau after approximately 10 minutes. The rate of EZ development for the other two buffers was considerably lower. Once formed, the exclusion zone tended to persist for many hours, and long-term observations showed that the region was stable for days.
Noteworthy is the observation that whether the nucleating surface was anionic or cationic, the EZ was seen, and was in fact approximately the same size. That is, negatively charged microspheres were excluded from a 200-μm zone around the bead, whether the bead’s polarity was negative or positive.
Several potential artifacts attendant with the formation of exclusion zones have previously been considered and tested [1
]. No reason could be found to suggest that the EZ was anything but a genuine feature. An additional control was carried out here, using exhausted beads, i.e., beads that had effectively lost their ion-exchange properties. This loss is detectable through an indicator dye incorporated by the manufacturer into the beads, which fades with diminishing exchange capacity. Initially, it was noted that when one of the colorless (exhausted) beads was placed in the experimental chamber, no exclusion could be seen. To confirm this connection, beads that showed exclusion were soaked for several hours in either 1M NaOH (H+ form bead) or 1N HCl (OH- form bead) to completely neutralize the free counter ions from the exchange resin. Again, the beads lost their color. When placed into the microsphere suspension, the negatively charged beads produced only a small EZ, while the positive beads produced no EZ at all — confirming that the presence of exclusion depends on the physical chemical state of the nucleator, and is not the product of some secondary effect such as microsphere settling [10
], colloidal void formation [13
], or convective flow [12
In addition to Imidazole, two other buffers were explored. We used TES and MOPS, buffers commonly employed in biological preparations, and tested them over physiologically relevant concentrations and broad pH range [8
]. Both buffers exhibited EZs; however, exclusion zones were smaller and developed more slowly than with Imidazole. Therefore, the Imidazole buffer was chosen for use in all subsequent studies.
The reason that Imidazole was more successful may lie in that molecule’s unique electronic properties. TES and MOPS are both aliphatic zwitterionic molecules; Imidazole on the other hand is aromatic. Furthermore, one nitrogen in Imidazole is part of the π-conjugate aryl ring, while the second nitrogen projects beyond this conjugate system and presents sigma-unpaired electrons. How this may play a role in the improved pH stability of the EZ is not entirely clear, but may indicate that the region of water adjacent to these surfaces has a novel electronic structure that may include conjugated or solvated electrons. Imidazole is also a weak chelating agent, and in previous work [16
] it was found that salt contaminants could diminish the EZ size. Therefore, experiments were conducted replacing buffer with EDTA, an effective chelator; an exclusion zone could be found around negative beads only, and the shape of the EZ was easily distorted. Thus, it is apparently not Imidazole’s chelating capacity that accounts for its stabilizing influence.
To explore the role of buffering capacity the concentration of Imidazole was varied from zero up to 50 mM. Results are shown in . EZ width increased sharply with increasing buffer concentration, reaching a maximum of 280 μm at 2.0 mM and 380 μm at 0.5 mM, respectively, for positively and negatively charged beads. Higher buffer concentrations diminished EZ width to stable values near 200 μm, the negative bead consistently exhibiting slightly larger exclusion zones than the positive one. Hence, for both types of bead, addition of buffer tended to stabilize EZ width to relatively large values.
Effect of Imidazole concentration on exclusion-zone size at pH 7.0. Error bars indicate degree of spherical symmetry within the EZ, i.e., shorter bars represent near perfect symmetry.
The EZ was not always spherical. At low buffer concentration (e.g. <5 mM) the EZ was typically distorted into elliptical or even teardrop shape, whereas at higher buffer concentrations (>10 mM) the exclusion region retained near-perfect spherical geometry. In , distortion is shown by the size of the error bars — smaller bars indicating more spherical symmetry. It was common to observe streaming behavior of microspheres in the surrounding bulk suspension, most likely driven by thermal convection. If the EZ distortion occurs as a result of streaming-induced shear, then the results imply that addition of buffer stabilizes the EZ, allowing it to retain its spherical shape.
Preparing Imidazole-buffered microsphere suspensions with pH values ranging between 3 and 9 tested the stabilizing influence of buffer on the exclusion zone. Solutions outside of this pH range quickly exhausted the ion-exchange capacity of the beads, which resulted in a collapse of the EZ or no EZ formation at all. In the absence of buffer the EZ is profoundly sensitive to solution pH, showing up to a 5-fold size variation [2
]. Addition of 10 mM Imidazole limited the mean size variation to ±30%.
To determine whether electrical features of the EZ are similar to those seen in the absence of buffer, potential measurements were carried out using glass microelectrodes. A measuring electrode was translated vertically downward towards the bead surface; the reference electrode was immersed within the same chamber many centimeters away from where the measurement was made. Electrical potential measured as a function of distance from the bead surface is plotted in . For both types of bead, the highest magnitude was found at the bead surface (~150 mV), cationic beads showing positive potential, anionic showing negative. The potential decayed steadily with distance from the bead surface, reaching zero at the outer edge of the EZ. This potential distribution is consistent with observations from earlier work with negatively charged surfaces in pure water only [2
To check for possible aberrant microsphere behavior, microsphere dynamics were observed under the influence of a purely coulombic force. Platinum electrodes were immersed into the buffered suspension and DC voltages between 250 mV and 5 V were applied. As anticipated, the negatively charged microspheres translated away from the cathode and accumulated around the anode. Switching polarity in situ resulted in immediate reversal of microsphere movement. Varying the applied voltage changed the microsphere migration speed proportionally; i.e., at higher voltages the microspheres moved more rapidly. Thus, microspheres situated in applied electric fields behaved entirely as expected. Nothing anomalous that might have contributed to the generation of exclusion zones could be found.