February 25, 2020
by Thomas Porter
Click-chemistry was used to conjugate DNA to secondary antibodies which allows for the reversible binding of fluorescent DNA cages. In this particular experiment, a DNA cage concentration of approximately 200 times the previous concentration was used. There is a bright signal from the samples, but also a large amount of non-specific binding (Figure 1). The images of the experiment seem to be brighter than the negative control, but the non-specific binding makes it hard to quantify.
Due to some limitations with acquiring more secondary antibodis a weaker signal due to the lack of amplification from the primary-secondary binding but also has less non-specific binding of the cages (figure 2).
Our next goals are to repeat the direct primary antibody conjugation and the secondary antibody conjugation at a lower concentration of the DNA cages as well as using a longer incubation time of the cages in the blocking buffer before conducting the cell labeling. This should help to reduce the non-specific binding problems so that the true signal can be observed better.
The following information is regarding quantum dot (QD)-DNA conjugates prepared with a phytochelatin-3 (PC3) coating with single-stranded DNA conjugated via copper free click chemistry.
I previously observed a decrease in fluorescence of QD-DNA conjugates stored in the dark at 4 °C over time (Fig 1).
I believed this was occurring by oxidation of the QDs. Because the PC3 surface coating is very thin, oxygen can easily diffuse across the coating and react with the QD. This results in decreased fluorescence. The concentration of these QDs was 3 nM. In the last bar (orange), I prepared a fresh dilution of QDs to see if the fluorescence decrease could be attributed to dilution only, instead of oxidation over time (I would expect a high fluorescence intensity for this bar if oxidation over time was not the cause/dilution over time was the cause).
To overcome this problem, I decided to synthesize a new batch and sparge it with argon (inert gas). In this process, argon is bubbled through the aqueous solution containing QDs. Oxygen initially dissolved in the solution diffuses into the argon bubbles being introduced to the solution and gets removed. I then kept two samples to determine the best storage conditions for maintaining QD fluorescence over time:
1. I took some of the sparged QD solution and stored it in the dark at 4 °C (Fig 2). This solution was not kept under argon flow after initial sparging.
2. I left some of the sparged solution under continuous argon flow in the dark at room temperature (Fig 3).
The key differences between these two samples are the temperature (4 °C vs. room temperature) and surrounding environment (air, which has oxygen that can diffuse into solution, vs. argon). These QDs were kept at ~120 nM. I attribute the variability observed in sample 2 to instrument variability of the fluorometer (which I have observed in other studies not shown here). From these data, it appears that sparging the QDs with argon and storing them under continuous argon flow is the best solution for maintaining fluorescence over time.
Currently, I am working on optimizing gold nanoparticle conjugation with single-stranded DNA. Future experiments will be focused on photoswitching using the optimized nanoparticle conjugates.