Tuesday, August 10, 2010

Mancuso: Summer Immersion Week VI

Week Six marked the largest progress in terms of research progress on my project. My thiol-modified oligonucleotides arrived and I was able to start making real progress building a new detection system for KSHV. Really two big tasks were left in front of me; attaching the oligonucleotides to the gold nanoparticles and then testing the system with viral DNA.

The first task didn’t appear too difficult, but as I began I realized it was going to be hard to detect whether or not my oligonucleotides attached to the gold nanoparticles. I had thought the added mass of the oligonucleotides would cause a shift in the SPR of the particles, but realized because my oligonucleotides were short this wasn’t going to be the case. The aggregation of gold particles would still work; I just needed another method of determining whether the first step of my preparation had completed.

Ultimately I came up with another test to determine whether the oligonucleotides attached to the gold nanoparticles. Specifically, gold nanoparticles react with salt and precipitate out of solution. However, if the surface of the particles is protected by oligonucleotides the particles remain stable in suspension. This stability is also particularly important because DNA hybridization is normally performed in the presence of salt, and will be required for the next step. By running an array of samples changing the concentration of salt and whether or not oligonucleotides had been bound to the gold nanoparticles I was able to confirm their presence. Looking at the figure the gold nanoparticles with no nucleotides rapidly precipitate out of suspension and the solution loses its color. However, once a monolayer of oligonucleotides is attached to the particles they remain in suspension for days.

The next step is to actually test these nanoparticles with DNA from cell lines infected with the virus and see if I can get a color change reaction. So far I’ve been unsuccessful, but I have another week to try. I think the problem might be the proximity of the gold nanoparticles; even though the lengths of DNA I chose closely match the literature I’m worried the particles are still too far. I have some particles with larger radii which I plan on trying which might help alleviate this problem.

This week I also visited the Emergency Room which so far has been one of my favorite places in the hospital. I really enjoyed the fast pace of and was able to see how quick, easy to use technology would really benefit the patients here. I continued in pathology too and observed more patients and cases. I also saw a mortality conference on one case I had followed and was really interested to see all of the information presented together. Another great week in New York!

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