At Biolin Scientific, several of us that have worked with QCM-D technology for a long time. Aware of the fact that it is easy to get stuck with your own subjective view of the world, I reached out to two of my colleagues, Fredrik Pettersson and Erik Nilebäck, both Senior application scientists, to get some new perspectives and insights on the method. In the interview, Fredrik and Erik shared some of their extensive knowledge on QCM-D, as well as their different experiences using this technology.
There are different ways to describe QCM-D, Fredrik says. It is a label-free method, i.e., it measures molecular interactions and molecule surface interactions without any labeling. You can also describe it as a mass sensor. The abbreviation QCM-D is short for Quartz Crystal Microbalance with Dissipation monitoring, so it is a balance for small masses. It measures mass bound to a specific surface. You can also measure the properties of the material bound to the surface. Essentially, QCM-D is a surface sensitive, time-resolved technology. The time resolution is one of the great advantages, since it allows you to follow processes in detail, Fredrik says.
QCM-D measures mass in the ng range, Erik says. It measures molecular layers, i.e., single molecular layers, and larger structures on top of that, up to a few microns. We can measure small molecules as well, and single layers of those, Fredrik adds.
QCM-D is an acoustic method, Fredrik continues. It uses a sound wave to probe. This can be compared to for example SPR and Ellipsometry, which are optical methods. The optical methods don’t measure mass, they measure changes in refractive index. The optical techniques don’t measure the coupled solvent, such as water, Erik adds. Also, with the optical technologies, you don’t measure structural rearrangements of molecular layers. You can see a change in the refractive index, but it is difficult to quantify, Erik says. Since QCM-D is an acoustic method, you can combine it with optical techniques to get complementary data about mass for example.
When I started as an application scientist, I thought we had quite complex and very much mathematics in our explanations of the fundamentals of the QCM-D, Fredrik says. This made me think a lot about the theory and how to describe it in a straightforward way. Yes, QCM-D is an acoustic method, and sound waves are what we are measuring. A vibration, i.e., a sound wave, is being transmitted through the layers and the liquid. You can say that the quartz crystal itself gives a tone. The QSense sensors have a fundamental frequency of 5MHz. This is ultrasound. Very, very high ultrasound. In contrast to medical ultrasonic measurements, we don’t measure time to the reflection, instead we measure how a layer on top of the quartz sensor itself affects the sound, the tone level. If you have a thicker layer on top of the sensor, you get a lower tone, just like a longer guitar string gives a lower tone, Fredrik explains. We measure frequency change that is directly related to mass. We also measure how the sensor gets dampened, i.e., how the oscillation dampens and dies out after you stop stimulating it. This gives you the material properties. In brief, it really has to do with how sound waves propagate through the film you are measuring and out into the liquid. And this gives us information on mass changes and structural changes with QCM-D, Erik adds.
I started using QCM-D during my master’s thesis and used it throughout my PhD project, Erik says. QCM-D was my main technique, and I used it for all kinds of biomolecular systems, from biotinylated systems such as biotinylated antibodies, biotinylated carbohydrate layers, and then looking at repeated antibody-antigen interaction, to carbohydrate layers looking at cellular interaction. For this system, I combined it with light microscopy.
I was mainly working with specificity, I wanted to measure specific interactions, he adds. We also looked at conformational changes in proteins, where we could measure the conformational changes and verify them with dissipation shifts. The conformational change was triggered with known drug molecules. When changing conformation, the molecules would get elongated, which could be detected with the dissipation signal. This was a novel way of looking at conformational changes for that system. In a similar fashion, with the carbohydrate system, we used enzymes that we knew would degrade these systems. There we could see the specificity and compare that in different systems. So, essentially the focus of my PhD work was to verify the biological activity of the layers on top of the QCM sensor, and to develop new strategies or protocols for using then technique, Erik says.
My first experiences of QCM were with thin film deposition, Fredrik says. There you have a vacuum system, and you sputter or thermally evaporate metals on top of Si wafers. For that purpose, you use the QCM calibrated just for the density of the metal and then you can get the deposition speed read out quite easily. Compared to QSense, these QCM instruments are quite crude. They have low resolution, and you measure very rigid layers. When I started at Biolin Scientific, I started working with development of the QSense instruments, Fredrik continues. We were on the brink to launch our automated instrument, QSense Pro, and there we had to work a lot with aspects like how does the instrument hold the sensor, how can we increase the reproducibility in sensor clamping, how can we make a flow cell that fills reproducibly by a robot every time, without any air bubble formation, and how can we keep the temperature control stable when the robot pipettes liquid. There were a lot of questions to consider to really take advantage of the precision and extreme resolution of the QCM-D method. This was my way into the company and my bridge over to application development, where I currently work. Now I work, for example, with development of QCM-D demo experiments. One important aspect when doing a demo at a customer site is to have sufficiently clean sensors, which can be a challenge when working in someone else’s lab, he continues. So, it has been close to my heart to identify where the limit is in terms of reproducibility. I have also been working with demo protocols which highlight the quality of the QCM-D data and shows that you see the change in the material properties together with the mass change. Trypzination, for example, is a cool experiment to do on a QCM-D sensor. There is so much information in such a measurement and the time resolution is so fast that we can follow the entire scenario. It is fascinating! It gives you so much detail on what’s happening at the surface, Fredrik says.
In general, QCM-D is useful when you are interested in looking at the molecular scale, Erik says. Especially if you are talking about interactions and you want to know how and when the interactions happen, i.e., if you need time-resolved information. If you know that the materials that you want to study are soft, if they have viscoelastic properties, like polymers or biomolecules, then the QCM-D technology is a perfect tool. You could also use QCM-D if you are interested in seeing how bulk materials interact with the surface. If you are interested in the details regarding what happens at the molecular level, the possibilities are, more or less, endless, Erik concludes. Fredrik agrees. And within the scope of biomolecular applications, you can for example look at protein aggregation, Fredrik says. In this case, one of the advantages is that you can follow the single molecule interactions with the surface, and then up to 10 000 times thicker layers, all the way up to um. It is quite outstanding to be able to follow events over such a wide size range; from aggregation onset seeing the first monolayer of interaction with the protein, to the dimerization and the multilayer formation and eventually the aggregation process, which eventually also leads to structural changes of the protein itself when you go to thick layers. The ability to follow such a wide size range with that precision is rather unique, Fredrik continues. This is an advantage in many, many applications. And of course, in contrast to many other surface sensitive techniques, we study the exact material surface. This allows you to measure the interaction between the liquid, the surface, and the solute being solved in the liquid. So, you can study all three components of your system, i.e., your molecule, your surface, and your solvent, using a huge range of different materials, which is quite unique. This is worth emphasizing, Erik adds. You are really free in the type of material that you use on the sensor. That is something that drives the applicability of QCM-D. The flexibility in terms of materials means that you can study phenomena in detail, Fredrik says. For example, you can study different formulations of a polymer and analyze the impact of different softeners, or different polymer chain lengths, and then you can follow in detail for example swelling behavior, polymer removal, or adsorption. In this sense, QCM-D is a very realistic method, where you work with real surfaces.
Listen to the interview to learn more about Fredrik’s and Erik’s experiences using the QCM-D technology, including a discussion on what information that the method offers, and a comparison of the pros and cons of different QCM:s. In the conversation we also talk about how QCM-D measurements are run in practice, and Fredrik and Erik share some useful tips and tricks on how to get the most out of this surface sensitive technology.
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