QD-dye conjugation!

March 29, 2018

by Abhilasha "Abby" Dehankar

By the last update, aqueous transfer of quantum dots (QDs) was optimized, wherein the native surface ligands of the organic QDs were replaced by peptides (Phytochelatin-3, PC3) that coated its surface in a loops-trains-tails fashion exposing carboxyl (COO-) and amine (NH2) groups of PC3 to the water. The surface charge of these QDs, studied using zeta potential, was effectively negative as PC3 constitutes of more carboxyl groups as compared to amine groups (3:1). These carboxyl groups could further be exploited to conjugate QDs to amine terminated single stranded DNA (ss-DNA) using the EDC-sulfo NHS chemistry as described in Figure 1. However, the conjugation to DNA turned out to be unsuccessful. The length of the DNA was proposed as a possible reason for the failed binding and to test this hypothesis a control experiment of QD-dye conjugation was proposed, as the size of DNA >> size of the dye. Simultaneously, QD-dye conjugation would also act as EDC-sulfo NHS chemistry functioning indicator for QD-PC3. Therefore, this report is focused on QD-dye conjugations.

EDC-sulfo NHS chemistry
Figure 1: EDC-sulfo NHS chemistry

First, carboxy-terminated QD-micelles were conjugated to an amine terminated dye using EDC-sulfo NHS chemistry. This experiment was successful confirming that our conjugation chemistry is operational. Next, the same chemistry was extended to QD-PC3 for conjugation with the dye and which also turned out to be successful (Figure 2). For both the experiments above, the dye:QD ratio used was very high (~3500). However, to extend this procedure to the expensive DNA, it is necessary to decrease the amount of required DNA. To confirm the conjugation at lower ratio, the same conjugation procedure was carried out at dye: QD ratio of (200:1). Unfortunately, this experiment was unsuccessful. The difference between a successful and failed batch demonstrated through fluorescence spectra in Figure 3. This indicated that maintaining a high ratio of target molecule to QD is extremely important. To allow the usage of lower amount of DNA, the target molecules for next reactions would consist of a mixture of amine terminated DNA and amine terminated filler molecules (for example: PEG-amine) to still maintain the required QD to target molecule ratio for successful conjugation.

Firgure 2: QD-micelle and QD-dye conjugation
Firgure 3: QD-dye conjugation fluorescence data

 

P.S.: One major change that contributed to better retention of successful conjugation was the use of column purification as opposed to centrifugal purification.