May 21, 2018
by Abhilasha "Abby" Dehankar
During our last phone call, we focused on understanding the conjugation of our peptide coated quantum dots (PC3-QDs) to amine terminated dye molecules. This study was performed with an aim to understand and optimize the carboiimide conjugation chemistry for our system while minimizing any kind of interactions with the target molecule. The experimental results indicated that a high target molecule to QD ratio is required for a successful conjugation. To make this process cost effective while maintaining a high target molecule to QD ratio, we decided to use a mixture of amine terminated polyethylene glycol (PEG-amine, the filler target) and amine terminated single stranded (ss) DNA (the actual target molecule) to achieve the desired ss-DNA conjugated QDs. In addition, to permit the detection of DNA conjugation to QDs in lab, the amine terminated DNA was ordered with a dye tagged to the other end of DNA (Figure 1). Following sections of this post are focused on conclusions from the aforementioned experiment.
While waiting for the ordered materials to arrive, we continued the optimization of the carboiimide chemistry on QD-dye conjugates by varying the conjugation parameters such as QD to EDC ratio, EDC: sulfo NHS ratio and so on. These experiments demonstrated that we can drastically reduce the required DNA to QD ratio (from about 1500 to 15) by significantly increasing the EDC to QD ratio! Figure 2 shows successful conjugation of dye molecules to QDs at a dye : QD = 1:15 and EDC : QD = 1:50,000.
In addition, for QD-dye conjugations, removal of excess/unconjugated dye molecules was accomplished using spin columns that were capable of separating molecules below the size of 2 kDa (size of dye< 1kDa). However, size of DNA for QD conjugation > 2 kDa. Therefore, we designed a new size separation column with desired capabilities. These separation columns were further tested on our QD-dye conjugates and were successful in separating the unconjugated dye molecules from the actual QD-dye conjugates.
Finally, on arrival DNA and PEG amine, the optimized conjugation chemistry was performed on QDs in presence of both DNA as well as PEG amine. Unfortunately, this experiment was not successful. Thus, it is evident that the nature of target molecule greatly affects the conjugation. This could be a result of steric or chemical interaction of target molecules with the QD surface or both. The interactions with QD surface can be greatly reduced by passivation of some of the carboxylic groups as well as amine groups on the surface of our QDs. This could be achieved by conjugating them with modified PEG amine and PEG carboxlic acids, respectively while maintaining their aqueous stability and followed by conjugation with DNA. Further, literature suggests minimal interaction of the nucleotide “thymine (T)” with charges on QDs and therefore future experiments would be conducted by using a DNA sequence consisting only of T nucleotides (PolyT). These experiments would definitely get us one step closer to answering the question: Does DNA sequence matter during QD- DNA conjugation?