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This week I’ve been spending a lot of time working with the Nano ITC, so I thought I’d take some time to explain a bit about how it works. After all, this is what I’ll be using all summer!

Thar she blows! The Nano ITC in all its glory!

The Nano ITC, which stands for Nano Isothermal Titration Calorimeter, is a fun little box that can tell us all sorts of things about chemical reactions. In understanding what this instrument does, I find it useful to break things down word by word. First up is “Nano,” which means it’s pretty small. In fact, the ITC only uses about 350 microliters of sample per trial. That’s about .0006 times the size of a Venti cappuccino at Starbucks! This lets us use a lot less sample to get the same information.

A view down the barrel of the ITC. At the bottom you can see the openings of the two chambers. The one in the center holds our sample and the one on the right holds our reference.

The second word in the name is “Isothermal,” literally meaning “same temperature,” which is exactly how the ITC works. Inside this box of wonder are two chambers, which are held at the same temperature throughout the experiment. As the temperature of our sample changes, it is checked against a reference solution placed in the other chamber, and the machine adjusts its temperature so the two match. The ITC measures the amount of power it takes to keep these two chambers at the same temperature, which gives us our results.

This is the syringe that the ITC uses to inject one of our solutions into the other.

The third word in the name is “Titration.” This is a fancy chemistry word that basically means we’re adding one chemical to another. The ITC uses a syringe to slowly inject small amounts of a solution into our sample. This causes a chemical reaction which we can collect data about. Also, we don’t actually have to do anything after we start the machine, since the ITC does it for us! Finally, we have “Calorimeter.” This just means that the ITC measures information about the thermodynamics of whatever chemical reaction is happening in the machine.
Putting all this information together, we see that the Nano ITC is a device that lets us measure thermodynamic data about a chemical reaction between two solutions. It does this by measuring the power it takes to keep our sample and a reference at the same temperature, and it doesn’t even need that much solution to do it! And there you have it! That’s a basic run-down of the Nano ITC. It’s a really powerful tool that can give us a ton of useful information. Hope you liked the explanation, and I’ll be back next week with some results from this week’s testing!

I would like to welcome Diana, Ben, and Nick, three wonderful new students to the lab. We have just begun our adventures together and already they are showing great promise. Keep checking back here for exciting developments as they tell us about their research!

So last week Monday morning came around and Professor Thompson had been too busy to prepare gold nano-particles for me. This was fine, I was ready to tackle the task on my own. The procedure involves bringing 100mL of a solution up to a boil and then pouring 3mL of another solution into it. The solution then proceeds to change colors several times, its actually pretty neat to watch. I ended up making a pretty concentrated batch of nano-particles. It even ended looking pretty good under the UV-Vis, so I decided to continue on with my project using them. I needed to find a good ratio of NPs and PAH because the two are pretty fragile. If not mixed together properly they can immediately aggregate and make the solution unusable. Also, if they are not in the proper ratio this can also cause it to become aggregated and then unusable. So I tried to make a 3x and a 5x diluted NPs solution with normal concentrations of PAH. Both seemed usable after mixing. I then tried out the new cleaning procedure that Professor Thompson had suggested. I would spin the solutions at very high speeds, 9000 rxg and 7000 rxg in order to get all of the excess PAH out of the solution. This maximized our risk of contamination by stray PAH particles. My problem with this procedure arose when I had to wash the pellets with DNA in them. After I started this centrifugal process, I found that the solutions were creating pellets that were virtually non-existent. This is not good, as it is one of our main goals to get good pellets here. I was thinking that these pellets could not form because I had diluted the initial amount of NPs in the beginning. Professor Thompson suggested it had something to do with my spinning procedure for the DNA washes. I’m now currently trying this new procedure, which includes longer times and a larger rxg, to get better pellets in my solutions.

The last run of samples was supposed to be our most promising samples yet. It had looked like we were going to get not only quality data but in a large quantity as well. Unfortunately, something had gone awry. When we start with out citrate nano-particles they should be negative in charge, then we wrap them in PAH and they switch to positive charge, and then finally we wrap them in DNA and they switch charges a final time, back to negative. My first two Zeta tests were conclusive and yielded the expected results, however the final one simply doubled the number and did not switch the charge at all. Professor Andresen phoned a friend and we then scheduled a conference with Professor Thompson. Before we could have this conference however, I first had to prepare a power point presentation on my most recent bit of research. Dr. Thompson broke down several of the data pieces I am so accustomed to collecting and helped me better understand them. The UV-Vis is a lot more helpful then I know it to be, but I really need to learn how to overlay graphs on it, I am determined to learn this early next week when I have to use the machine again. The conference left me feeling very hopeful about the project. On Monday Professor Thompson is going to prepare more nanoparticles for me to use in my next batch of samples. He also suggested a much more appropriate way of preparing the PAH wrap and how to more properly purify them. I am hopeful that this will greatly improve the project. At the end of this week, I am wrapping it up by shearing DNA and looking forward to finally getting proper data next week.

So at my last post I had just found an appropriate mix of salts and nano-particles. This allowed me to then mix in my DNA and start performing equilibrium dialysis. However, I immediately ran into another big problem. What was I to re hydrate my solutions with? In the past, at this step I was not using citrate nanoparticles, but I was re hydrating with TEM buffer. I was uncertain if this would be efficient with these new particles. I decided to head to the science center and discus this problem with Rich, a fellow user of similar nano-particles. We threw a few ideas back in forth, but nothing seemed like an exceptionally good idea to re hydrate with. So I had to make a sacrifice, I gave up any hope of getting to use the ICP-OES this run in order to figure out what to re hydrate my samples with. I split my total solution of nano particles into three parts, one would be re hydrated  with mili-Q, another with TEM buffer, and a third with a solution that was 10^-2 M NaCl. Unfortunately I was only re hydrating with 4mL each when I typically do double that. Regardless, the results appeared to be conclusive, showing that the salt buffer worked the best.

So then I had to restart once again. This time, I took all of my gold nano particles and coated them with PAH. I then spun them in the centrifuge for an increasingly long period of time to siphon out the supernatant and continue spinning that. Finally, I recombined the pellets and diluted it back to 7mL with more nano particles. So I’m left with 1 super concentrated gold nano particle sample and 7 other slightly less concentrated samples. They were then all mixed with DNA and left to sit over night. Now I am currently trying to perform equilibrium dialysis, and hopefully after doing this I’ll finally be able to use the ICP-OES.

Wow! it’s the sixth week, seems like time never stops and always keeps moving. Well today is a Monday, which are never a good feeling but we gotta hang in there. I started my day by going over the results with Professor Andresen, in order to come out with a solution or a new procedure. Eventually professor Andresen did came out with a new procedure which is the same procedure has before but with slightly different trial digestion. Instead of creating different samples of our solution by adding different amounts of micrococcal nuclease, we instead only made two  50 micro-liters of solution which contain different amounts of micrococcal nuclease. I places this solutions into the heater for 37 degrees Celsius and took out 5 micro-liters of each solution at a specific time. The different times that we assigned were 5, 20, and 40 minutes, with another sample that lasted about 2 hours. This procedure accomplished the task of creating different sizes of DNA  by letting the microccocal nuclease eat up the nucleic acids and taking small amounts of sample at different times and stopping the process by adding EDTA. Once this process was finish, I continued the trial digestion with the same procedure as before by using proteinase K and SDS. In the mean while, I created a new gel in order to put the trials in tomorrow. I also made more TBE buffer to used the next day. All this procedure took approximately the whole day due to the unfortunately broken wrist I got. All is well and I could still work, and that’s really important to me because I never give up on something I start.