August 1, 2018
by Kil Ho Lee
In these experiments, we are attempting to create QDs conjugated to DNA using a procedure known as “DNA embedding.” In this procedure, organic QDs are transferred into the aqueous phase. Then, a ZnS shell is grown on their surfaces. Some thiolated (-SH) DNA is added as a partial sulfur source, which permits molecules to directly embed or grow with the surface crystal. This is different from chemical conjugation to the surface, as the molecules are embedded in an interior crystal layer.
Previously, we employed ligand exchange to transfer QDs into the aqueous phase. We found that 3-Mercaptopropionic Acid (3-MPA) is a suitable for transfer from organic (i.e. chloroform, hexane) phases to the aqueous (i.e. water) phase. Our previous reports described process optimization to successfully replace the original octadecylamine (ODA) ligands on the QD surface with 3-MPA. This optimized process worked very well for red QDs (~ 610 nm emission), but not as well for green QDs. Green QDs are required to create the on/off switching needed for STORM imaging because the green light overlaps with the absorbance of the gold quencher. This general finding may stem from the higher surface to volume ratio of green QDs compared to red QDs, as green QDs are smaller. The surface of a nanoparticle is the least stable region and is chemically active to external species, which can cause a loss of fluorescence or water solubility. Thus, using water-soluble red QDs as a temporary model, DNA embedding steps were explored to determine if the process works.
Since then, a new challenge emerged that further impeded optimization of the DNA embedding technique. The previously optimized ligand exchange technique has not been working with a new batch of QDs. Thus, our process is not as robust as will be required for future uses. Hence, in this report, we took a step back and attempted to optimize our aqueous transfer procedures to make them more robust.
Briefly, we achieved successful outcomes using the same ratio of 3-MPA to QDs (or more specifically to the ODA on QDs) following the same experimental procedure, except we changed the organic solvent used to initially disperse the QDs. Previously, hexane was used as an organic solvent to disperse QDs. Then, 3-MPA containing water (pH 10) was introduced. Based on our experience, hexane was employed because it had previously promoted better long term QD stability compared to chloroform. However, given our issues with repeatability of procedures, we attempted ligand exchange using chloroform as the organic solvent (Figure 1).
Figure 1. Ligand Exchange to replace ODA on QDs (Left: Green, Right: Red) with 3-MPA
After purification, the fluorescence intensity of 3-MPA coated QDs was compared to that in the original organic solvent or chloroform (Figure 2). Both green and red color QDs, displayed high fluorescence intensity; for imaging, the fluorescence intensity above ~ 105 1/s is recommended. Green QDs, however, yielded an interesting result in that the fluorescence intensity for ligand exchanged aqueous QDs was higher than that of the original, organic QDs. This is rarely observed and the mechanism leading to this result is not clear yet. The opposite trend was found for red color QDs. However, direct comparison was not possible because at the absorbance matched concentration for organic QDs, the fluorescence intensity was too high and the photo-detector was saturated as shown by the flatten curve near the emission wavelength (600 nm) in Figure 2.These data indicate that these optimized steps worked very well for both green and red QDs because they retained high fluorescence in aqueous phase.
Figure 2. Absorbance (Abs) and fluorescence (Fluo) spectra of QDs before (organic: org) and after (Aqueous: Aq) ligand exchange.
Ligand exchange worked better when QDs were initially dispersed in chloroform. This may be because of the solvent compatibility of ODA coated QDs in hexane versus in chloroform. In fact, based on Hensen solubility parameters (HSP) of ODA, chloroform, and hexane supports the higher solubility of ODA in chloroform than in hexane. Specifically, because ODA is more soluble in chloroform, replacing ODA in chloroform with 3-MPA is expected to be done more effectively. The next step is to use aqueous green QDs for DNA embedding.
 Larsen, M. (2009). Hansen solubility parameters and SWCNT Composites. In 17th International Conference on Composite Materials (ICCM-17).