Discussion
Why isn't the background crystal clear?
As seen in TEM results, we can notice that there are some unknown debris scattered on the background. This is actually due to the limit of purifying technique in our experiment. Centrifugal filters were used to purify the assembled components. In official guideline, they suggest 14000 g as the best centrifuge parameter. However, our products seem to be too fragile to withstand the centrifugal force. When under the condition of 14000 g, we could hardly observe clear images of our products. This may result from the reason that they were shaken so hard that the structure was damaged. In order to preserve the structure's integrity, we reduced the spinning force to 7000 g when filtering. At last, we observed our desired products; however, due to the mild centrifugal force, there were some debris left in the mixture. Hence, it is a trade-off between a clear background or a relatively clear image of our product.
Why isn't there a picture of both lids flipping up?
The answer to this question may also lie in the purifying technique. With much debris in the mixture, it may hinder the lids from flipping up since the motion of the lids utilizes Brownian motion only. More debris means more obstacles for the lids to move.
How to improve the purifying process?
It seems that the purification process is the major problem in our project, and it is crucial to improve the efficiency of this process. In the future, we will try gel purification, PEG precipitation, or gel extraction to optimize our purification results. This way, we may be able to solve the two problems mentioned before.
Further validation?
In TEM results, we successfully observed each of the lids flipping up. It is obvious that the lid is indeed triggered one side at a time because there's only one flipping lid when we added a single signal into the mixture. In the future, we are going to conduct various experiments to provide more solid proofs. For example, we can utilize FRET (Förster resonance energy transfer) to observe fluorescence changes.
While TEM provides us with clues on whether or not our device flip, using FRET allows us to check the stability of our flipping mechanism by telling us if the distance between materials are close enough for reaction to take place. Therefore, with the help of TEM image and FRET, we can know that our device can actually flip, and that the result of this flip, which brings materials closer, can lead to desired reaction.
As shown in figures below, green fluorophore is the acceptor of blue fluorophore and the donor of red fluorophore. By stimulating the blue fluorophore, we can detect emission of spectrum to verify the flipping state.
Flip the left lid &
emit green fluorescence
Flip both lids &
emit green and red fluorescence
Flip the right lid &
emit red fluorescence