Analyzing DNA-mercury Nanoparticles with Scanning electron Microscopy

 

Analyzing DNA-mercury Nanoparticles with Scanning electron Microscopy

In the last post I discussed how I used fluorometry experiments to investigate the binding dynamics of DNA and mercury ions. In this post, I'm going to talk about how we visualized the DNA - mercury samples via Scanning Electron Microscopy (SEM) to get more information on the structure and morphology of the particles formed by these interactions. As they say, a picture is worth a thousand words!

Remember how I said that the DNA forms some kind of hairpin or secondary structure around the mercury ions? Well using SEM we were able to visualize these structures more closely and see how they behave!

How did I Prepare the Samples for Visualization?

These samples, especially samples containing biological material like DNA, cannot just be thrown into the microscope and visualized. If that happened, the DNA would actually be invisible because of its inability to conduct electrons, since the conductivity of electrons is what actually corresponds to an image. So how did I prepare the samples so they could be visualized?

The first thing I did was to create the samples themselves. I had two control samples which were a sample with just the single-stranded DNA and a sample with just the mercury. Finally, the experimental sample contained both DNA and mercury. 

Next, I drop cast 10-20 microliters of each sample onto silica wafers, which are pretty much super thin strips of silica that many molecules are able to adhere onto easily. The wafers were adhered to the actual microscope stub via carbon tape. The samples were left to air-dry in a closed environment for 48 hours to allow the DNA - mercury nanoparticles to adhere to the silica.

At this point, the samples were dehydrated via a gradient ethanol dehydration. This just means that the samples ran through solutions of increasing percentages of ethanol (30% ethanol, 50%, 70%, and so on). This takes any additional water out of the system since water can really affect SEM readings. After the gradient dehydration, the samples were dried for another 24 hours.

Finally, at this point, they were almost ready to be inserted into the instrument. But one more thing had to be done. In order for the DNA to actually be visible, a coating of conductive material must be applied to the sample so that electrons can conduct through the particles so they show up in the image. This coating, called a sputter coat, was a thin, 5nm-thick coating of gold and palladium. Very expensive stuff! Once the coating was applied, the samples were finally ready to be analyzed!

The Results

The results we got from the analyses were amazing. To begin with, we saw nothing of note in the control samples. That was a good sign. Then, when we analyzed the experimental sample, we were able to get many images of what were presumably nanoparticles composed of DNA and mercury! 

What we primarily saw were a very wide distribution of particles and large aggregates. We clearly saw both single monomeric particles and very large aggregates/polymers composed of these monomeric subunits. These subunits were consistently small (between 25 and 75nm) and spherical.

Why is this important? It clearly demonstrates that the DNA is forming a secondary structure around the mercury ions. Observationally, it's doing this through both intramolecular DNA/mercury bonds (possibly in the case of the spherical monomers) and intermolecular DNA/mercury bonds (as in the case of the crosslinked aggregates where many of the monomers clump and link together).

Finally, this also confirms why Dynamic Light Scattering was such a pain for me and Dr. Wadkins in the beginning of this project. It's because there is currently no way to constrain the size of the nanoparticles to a certain range in solution. This would result in a nice clean reading from the DLS instrument. However, since the particles seem to be crosslinking in an uncontrolled manner, there's no size constraint and therefore a very wide distribution of particle sizes, leading to messy readings from DLS.

Below is a nice example of an aggregate of DNA - mercury nanoparticles that we observed during our analysis. You can see how it's made up of many smaller spherical nanoparticles:

Where to go from Here

At this point, my time at the University of Mississippi was nearing an end (at the time of this writing I've been home for about 3 weeks so I got a little behind on writing the posts). But I do have one more post before I conclude this series. In the next post I'm going to describe in detail my data analysis/ machine learning methods I used to help me draw conclusions from my Dynamic Light Scattering data and how those conclusions further support what we saw here with the SEM images.

As always, thank you for reading and I'll see you on the next one!

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