The range of technologies available for surface analysis, characterization of molecule-surface interaction and interfacial layers is vast. So, what analytical instrumentation should you use for your research? We talked to Dr. Jenny Malmstrom, Senior Lecturer in Chemical and Materials Engineering at the University of Auckland to learn more about what technologies that are used in her lab.
Our research is essentially about interfaces, Dr. Malmström says. It is about interfaces between disciplines, and physical interfaces. We work a lot with making engineered interfaces, to try to better understand, and in the next step, control biology, in particular stem cell biology, she says. We are also interested in using biology as an inspiration to template or design advanced materials.
On the side of understanding biology, we develop model systems to try to pinpoint some specific biological function that we would like to understand better, Dr. Malmström says. And on the side of using biology as an inspiration for advanced materials, we are interested to try to pattern magnets by using principles of self-assembly, or to hijack biomolecules or proteins for something else than their day-job. For example, some proteins are piezoelectric, and we are interested in seeing what we can use that for in the future. So, a lot of fundamental research, aiming to do something hopefully a bit more applied in the future, she says.
In one of our projects, we use an experimental model system where we are looking at both growth factor release, i.e., how growth factors signal in cells, and the process of mechanotransduction, i.e., how cells sense the mechanical properties of their surroundings, she says. To do that, we make a thin block copolymer film on gold. Along with the block copolymers, we self-assemble proteins, so the proteins go into one of the domains of the block copolymer film. In the future, that should be a growth factor, but at the moment it is just a very sturdy model protein that we can measure the release of, she says. On top of the block copolymer film, we put another layer to control and slow down the release of protein. And then, on top of that, cells will come in the next step to release the growth factor in a controlled way. The end goal is to make a surface where the cells must pull on the surface to release the growth factors, Dr. Malmström explains. And that would allow us to study the synergies between mechanotransduction and growth factor signaling.
Another project that we have been working quite a lot on recently is hydrogels, again for understanding cell biology, she says. We have tried to make hydrogels where we can control the viscoelastic properties. Often synthetic hydrogels are essentially purely elastic, so we have dialed in a second component to make them more viscous in a controlled way, Dr. Malmström says. We have also worked with hydrogels where we have tried to control the stiffness over time; we have made conductive hydrogels and then applied a potential to change the hydrogel properties. This project is also related to the questions around mechanotransduction, i.e., how cells sense their environment. A lot is known about how cells respond to stiffness, whereas less is known about how cells respond to the viscous ques. There is still a lot to find out there, she says. Also, there are not that many materials where you can change the stiffness on que so to speak, so that’s why we have been interested in making a hydrogel where we can change the mechanical properties by applying potential.
We work quite collaboratively, and use instruments all over the place, both at the university and more widely in the country, Dr. Malmström says. One of the methods that we use the most is AFM, and different modes of AFM, i.e., functional modes that you can use to look at the magnetic properties across the surface or the piezoelectric properties, etc. For surface characterization, we use contact angle instruments, XPS, QCM-D, Raman, and FTIR. For the gels, there is a lot of mechanical testing, such as tensile and compression, she says. We do a lot of AFM mechanical testing as well. And rheology. These are some of the workhorses.
XPS is a UHV technique, and we often use it at several stages, Dr. Malmström explains. It is one of those techniques that requires a slightly, higher skill to use. Our instrument is run by a technician, and you must book a time etc., so we only use it once we think we know what is going on. At that stage, it hopefully confirms what we think we have. If it doesn’t, we go back to the drawing board, she says. A typical example would for instance be the project with the protein inside the block copolymer. We wanted to show that we really had proteins inside, so we put our sample in the XPS. We couldn’t detect any protein because it was hiding inside the film, and the XPS is very surface sensitive. Then we leached the protein out and measured it again, and then we could in fact detect the protein on the surface.
There is a whole raft of functional modes in AFM that you can do, Dr. Malmström explains. In a standard measurement, i.e., if you just image in AFM, you tap along your topography and detect the topography a long a line. In most of the extra functional modes, a second pass over is added. There, you follow the topography at a set height, i.e., you are not in contact with the surface. So, say that you move up 50 nm or something, then you are sensing the forces at that range, for example magnetic forces or electrostatic forces. To measure magnetic interactions, you would have to have a magnetic tip, and to measure electrostatic interactions you would have to have a conductive tip and you apply a bias between the tip and the sample. The piezoelectric AFM is a bit more complicated; it is a contact mode. You resonate the cantilever in contact with the sample and you look at a contact resonance and how that changes, she explains. It can be tricky, and that’s part of the issue I think with using piezoelectric force microscopy on proteins because they obviously are not always robust enough for those contact interactions, and the piezoelectric response from a protein is frightfully small. So, it is hard to know what you are really getting out of an AFM measurement when it comes to piezoelectric force microscopy on proteins. But for magnetic properties and electrostatic properties, it is more straightforward. You are getting both the topography by doing one scan, and then the next scan you get those longer-range interactions, and you can then overlay those two images to really correlate what is going on in your sample, Dr. Malmström explains.
Contact angle instrumentation is easy to access for most people and is something that you can use for preliminary investigations of surface chemistry, before the XPS, she says. For example, we evaluated a lot of different surface chemistries to see if the block copolymer would assemble on anything else than gold. In that exploration, we would have done a lot of contact angle to see if we deposited what we thought we deposited on our various surfaces. We were also doing some layer-by-layer assembly on top of this block copolymer film, and then we would do contact angle to verify that the chemistry was changing between the different layers that we were building up, Dr. Malmström says.
We use QCM-D for example for the layer-by-layer systems to verify that things are building, Dr. Malmström says. Often, we combine QCM-D with imaging. If you are doing AFM, you just see the top surface so then we use QCM-D to try to verify how the layers are building in real time. We are not depositing the layers in a similar way when we are making the sample, however. For the AFM, we are spin-coating to deposit, so there is a discrepancy between what the sample really looks like and what we can look at with the QCM-D, so they are quite complementary. We also use QCM-D to look at different protein interactions at surfaces, enzymatic degradation of protein and so on, she says.
Another method that we use to measure the release of molecules from the block copolymer film is fluorescence spectroscopy. With this method, you tag the protein and then you can capture the release. Light based methods are useful in that way, she says. Typically, we would measure the fluorescence every two minutes, to capture the release event.
We also do mechanical testing and rheology, in particular on the hydrogel projects because that is quite central, Dr. Malmström says. The hydrogels are too thick to attack with the QCM-D, so we have had to use other methods. Often, we make the hydrogels macroscopic. They could be 200 μm or so, which is thick on a QCM-D standard. Here, we do quite a lot with the AFM, where you can do a force distance curve. It doesn’t work for all hydrogels though. Sometimes, they are simply too sticky, so you get stuck even if you do these measurements in liquid. For bigger gels, we do tensile testing, where you make a dog bone shape and then you pull it. We also do compression testing, which is the same principle as with an AFM, but you are not using a cantilever, so you are applying much larger forces, Dr. Malmström explains
The methods discussed so far are the methods that we use often, she says. In addition to these methods, there are other methods that we use now and then. For example, scattering on the beamline. We can do neutron scattering and x-ray scattering on the beamlines in Australia. Mainly, we scatter through the gels to get structural information about what is scattering and what size range that is. It is quite interesting to use both x-rays and neutrons because they interact with the material in different ways. We have also done a bit of magnetic measurements with the neutrons so polarized neutron reflectometry, where you are looking at the reflection of the neutrons from the surface, Dr. Malmström says.
Listen to the full interview with Dr. Malmström to learn more about her research, the instrumentation that is used in her lab, and her advice on which equipment to prioritize to invest in if the funding available for new instrument purchase is limited.
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Malin graduated in engineering physics in 2006, where her research focused on the QCM-D technology. Since then, she has been scrutinizing the how’s and why’s of the world in general, and the world of QCM-D in particular.