One of our featured topics for Diagnostics Weekend at Lablinn is Nanodiagnostics. I got into science by doing a research project in nanodiagnostics, so I think it's pretty apt.
I'm going to talk a little about the research project and then about some of the other cool nanodiagnostics things being done in an excerpt from my heretofore unpublished project write-up.
The Research Project
My 2015 BT Young Scientist project aimed 'to develop and functionalise a graphene-based biosensor to detect attractin, a protein biomarker of high-grade gliomas'. A mouthful, I know. For the exhibition booklet we had to fill in a 'title' and 'description' field and I actually just put that in for both.
In my project, I made a graphene field-effect transistor that could detect the presence of the protein attractin in a sample. Here's a quick diagram of it:
Basically, for that project I worked in CRANN in Trinity with one lab day a week for 8 months as well as 4 months of preparation, under the mentorship of a PhD student, a post-doc and a PI there, plus some lovely mentors in St. James' Hospital after that. I was interested in sensors and diagnostics and thought nanotech was cool, and after watching a documentary about Lilly Mae, an Irish girl with neuroblastoma, wanted to make a sensor for brain cancer because it's currently super invasive to diagnose. Obviously medical devices are complicated and require a huge amount of testing so it wasn't going to happen immediately on completion of the project, but I wanted to have a go at it and see how the idea worked in case it could be useful to anyone in future.
The goal was to make a sensor out of graphene, which is essentially a very thin ("2D") sheet of carbon atoms, in my case a one-atom thick monolayer, and then functionalise that using a combination of chemicals to make it detect something that indicated the disease was present (antibody), a linker chemical to help the antibody stay on the graphene (linker) and something to stop non-specific binding i.e. prevent false positives. I read a lot of papers for a few months to get myself reasonably up to speed and find a biomarker, i.e. a protein I could buy an antibody to detect that was present or elevated when someone had the disease. So in my case, having decided to make a sensor for glioblastoma because when I went home after watching that documentary I looked up 'what's the worst brain cancer' and it said that one was the most aggressive, I read a lot of papers that had studied what proteins were associated with the onset of glioblastoma, made lots of Venn diagrams and eventually settled on attractin, because I read that it was pretty much undetectable in the cerebrospinal fluid in a healthy person but rose sharply in a person with a glioma and rose even more with each cancer grade (grade IV is glioblastoma). As well as that, the paper said that attractin seemed to actually mediate glioma cell migration, so it definitely seemed like a good target. So basically, detectable attractin -> glioma.
I used anti-attractin antibodies, i.e. antibodies that would bind to attractin, because when a sample of cerebrospinal fluid containing attractin was applied to the sensor (which had electrodes on it), the antibody would bind, changing the conductance of the graphene which, thanks to the electrodes, could be seen on the electrical readout from the probe station I had set up next to it.
So once I'd done all the reading and after a lot of cold-emailing had gotten into a lab, I spent the next 8 months trying to get all the pieces together. First I needed to make monolayer graphene, and once I'd gotten that down (with lots of help from my PhD student supervisor Sinéad), I had to get a linker chemical to bind to it without breaking it, which was not easy. You may have read about graphene being an incredibly strong wonder material, but oh man it actually just kept breaking with the linker chemical I was trying to use, pyrene. Sinéad suggested switching to perylene, which also worked but didn't tear the graphene, so that was good and that's what I used from then on.
Antibodies and proteins are VERY EXPENSIVE (like, 500 euro for 0.1 ml expensive), so I couldn't use them for all the testing and used biotin and streptavidin instead, which are a common binding pair used for this because they bind very tightly to each other and can thus sort of represent antibody binding. So next I practised putting biotin on samples. Then I needed to figure out a way of stopping other things apart from the attractin/streptavidin binding and causing false positives, so I was advised to use Tween-20 and then figured out by experiment which durations-of-soaking-in-Tween-20 and concentrations-of-Tween-20 worked best for preventing non-specific binding without damaging the fragile, fragile graphene. Then, before adding the streptavidin (for the months of practise runs) or the attractin (for the final tests), I needed a way to deliver the sample to the sensor. At first Sinéad and I just dripped it on, but for the later tests we made a microfluidic cell and connected tubing to make it more precise. The first half of the process involved lots of Raman scanning to identify what had bound to the graphene and how pure it was, while the second involved lots of electrical measurements on the probe station to see if the electrodes were working properly or if there was leakage, and see if the attractin binding at relevant concentrations did in fact create a change in the readout from the probe station.
The good news? It did, at one of the concentrations. The bad news? Due to a bunch of unfortunate situations, the project had to stop after only a few of the attractin tests had been done, so I couldn't validate the sensor more or test it with actual cerebrospinal fluid samples and work out the issues there. I had many many streptavidin tests and Raman scans and electrical scans of various stages of the process, but only a couple of scans with attractin, which was a pity.
There was lots more to the process too, like cutting the silicon substrates with a very cool diamond scribe (like a pen with a diamond at the tip), the process of growing the graphene (in a furnace at 1350 C) and the preparations for that, and more. Thanks to my mentors and supervisors at the labs in CRANN and St. James' Hospital, and to my school and Frances O' Regan for being incredibly helpful with the project as a whole.
Now, read on for an intro to some of the research that'd been done before mine in an excerpt from my project's introduction.