Compact coating to DNA conjugation

February 20, 2018

by Abhilasha "Abby" Dehankar

The aqueous transfer is working very well!

Figure 1: Fluorescence of QDs transferred into water using the three component (pyridine-water-TMAOH) system (left) and two component (pyridine-TMAOH) system (right)

During the January update, I reported the very first successful “loops-trains-tails” aqueous transfer and low pH (pH 5) buffer exchange of organic quantum dots in our lab. However, this process was accompanied with a loss of fluorescence. Additionally, the reproducibility of the procedure was yet to be tested. Therefore, this update is focused towards achievement of efficient and reproducible aqueous transfer.

Firgure 2: Structural configuration of PC3 on QD surface in water

Generally, for aqueous transfer, QDs in pyridine are mixed with excess phytochelatin-3 (PC3) in water, followed by addition of Tetramethylammonium hydroxide (TMAOH) to increase the pH of the solution. This leads to a spontaneous ligand exchange and precipitation of QDs from the organic solution. The resulting QDs are therefore highly soluble in water.

  A couple of replicates of the procedure above lead to phase separation of the three component Pyridine – Water –TMAOH mixture along with precipitation of QDs and poor solubility of QDs in water. To avoid the interference of these phase separations to our aqueous transfer, the system was converted to a two component system of completely     miscible solvents Pyridine and TMAOH. This was feasible because of the high solubility of Phytochelatin-3 in Pyridine. The modified procedure turned out to be extremely reproducible and efficient with minimal (~20%) loss in fluorescence during the complete aqueous transfer.

For DNA conjugation, it is essential that Phytochelatin-3 takes a structural configuration in which the carboxyl and amine groups are exposed to the aqueous environment, while the thiol anion binds with the surface (Figure 2). Carboxyl group at any pH above 2 donates a proton and exists in the form of an anion that can be detected determining the “Zeta Potential” of the surface. A negative zeta potential (-4mV) was observed on the surface of the phytochelatin-3 coated particles indicating the availability of carboxyl groups for DNA conjugation.

Further, the DNA conjugation of these QDs was performed using the standard EDC-sulfo NHS chemistry with strictly controlled reaction conditions. The QDs exhibited extreme stability with minimal fluorescence loss during the whole process. The particles were also stable in the buffer conditions required for DNA origami. However, they did not show any specific conjugation to DNA origami hinges (Firgure 3). This observation was also accompanied with ~80% recovery of DNA after conjugation. Both the results indicate unsuccessful conjugation. My current hypothesis is that the DNA conjugation was not successful because of the length of current DNA. This hypothesis roots from the ongoing challenges we are facing in conjugation of long DNA (~20 base pairs (bp)) to gold nanoparticles. Our current DNA for quantum dots is also 20 bp in length. In order to test this hypothesis, an amine terminated fluorescent dye would be tested for conjugation with quantum dots using the exact procedure. Based on these results, either the DNA or the conjugation process will be revised.

Figure 3: QD-DNA origami composites