QCM-D technology is a powerful bioanalytical tool that is today widely used to study biological phenomena. It is for example used to study lipid membrane interfaces and has shown promising applications in pharmaceutical drug development and antimicrobial testing. In a recent webinar, Prof. Jackman, Associate Professor in the School of Chemical Engineering and Translational Nanobioscience Research Center at Sungkyunkwan University in South Korea, shared his experience using QCM-D in the field of membrane biophysics.
There have been a lot of exciting developments for QCM over the past couple of decades, Prof. Jackman says. One of the main areas where QCM technology started was looking at thin film deposition for semiconductors, optoelectronics and OLED displays. In this original format, QCM technology was mainly focused on looking at deposition of thin films in cases where the Sauerbrey equation could convert changes from resonance frequency into the thickness of nanometer scale films often used for vacuum deposition. Over the past several decades, led by the pioneering work of Prof. Bengt Kasemo and his research group at Chalmers University of Technology, there was huge innovation in QCM-D technology, really shifting from the QCM to QCM-D - Quartz Crystal Microbalance with Dissipation monitoring, Prof. Jackman says.
One of the greatest advances was developing QCM instrumentation where you can simultaneously measure resonance frequency changes, Δf, and energy dissipation changes, ΔD, at multiple overtones. Prof. Jackman says. One of the most powerful aspects of that type of advanced QCM technology was extending QCM from vacuum phase deposition to soft matter in biological applications, i.e., biological materials that are more viscoelastic in nature and where you will have an overtone dependent measurement response. The power of QCM-D technology is the ability to capture these phenomena. The ability to apply different types of analytical models to analyze the data has expanded the use of QCM-D technology to study various types of soft matter, including polymers, biomaterials, colloids and many more systems, Prof. Jackman says.
One of the most pioneering papers related to QCM-D technology was Prof. Kasemo’s paper back in 1998 [1], Prof. Jackman says. In this study, they looked at surface specific lipid adsorption kinetics at different types of surfaces, such as hydrophilic and hydrophobic surfaces. What QCM-D technology enabled, which was unique compared to other types of mass sensing devices at the time, was to look at not only the amount of lipid mass adsorbed to the surface, but the specific properties of how the lipid molecules, in this case, in the form of lipid vesicles, adsorbed to the surface, Prof. Jackman explains.
On surfaces like hydrophobic coated gold surfaces, you could see a monolayer absorbed directly. And on silica, vesicles absorbed until reaching a critical surface coverage before rupturing to form a bilayer. On oxidized gold, on the other hand, vesicles absorbed without rupturing. It is really this combination of simultaneous frequency and dissipation measurements that allowed discrimination of not only the total mass of lipids adsorbed, and lipid adsorption kinetics, but also what is the physical nature of the type of lipid assemblies that formed on the surfaces, Prof. Jackman says.
More recently, my mentor Prof. Nam-Joon Cho then at Stanford University extended this work to look at vesicle and bilayer structural transformations using QCM-D technology to study, for example, membrane-peptide interactions [2, 3]. So, this has been really powerful, not only to study fundamental biophysics of vesicle-surface interactions, but also to study more complex systems such as membrane-peptide interactions, Prof. Jackman says.
To build on those exciting early studies in the field, I would like to take an example of how the QCM-D technology is enabling new applications in the biotechnology and biophysics space, Prof. Jackman continues. One area where QCM-D has been used is in the area of antibiotic resistance. Antibiotic resistance is a huge global problem these days. Over the past century, antibiotics revolutionized health care, but there have also been problems with overuse and antibiotic resistance is becoming an increasingly serious health threat. This has prompted searches for antibacterial materials that work with different mechanisms than traditional antibiotics.
One area that we have been particularly interested in is called antimicrobial lipids. These are widely found in nature, and they are part of the innate immune system. These lipids are for example found on skin and in milk of many species. One of the most interesting things about antimicrobial lipids is that they have broad spectrum antimicrobial activity against enveloped viruses and bacteria. They work by disrupting the membranes surrounding viruses and bacteria. If you damage the coating of these viruses and bacteria, you impair their biological functions and inhibit their pathogenicity. Among the different types of antimicrobial lipids that we have been interested in are fatty acids and monoglycerides. The prototypical ones that we have often studied in this kind of model system include lauric acid and monoglycerides. These are molecules that are relatively similar in size and molecular weight, but they have very distinct membrane interactions which were revealed by the QCM-D technology for the first time, Prof. Jackman says.
Before our QCM-D studies looking at the membrane interactions, what was known beforehand was that these types of compounds have anti-infective activity, Prof. Jackman says. So, for example, if you treated bacterial cells with these types of compounds, they would cause membrane lysis. They have also been shown to have immunomodulatory activity, so, if you treated immune cells with these compounds, they could cause structural changes. However, prior to our studies there was very limited understanding of the interaction mechanisms of how these compounds work. It was often thought that monoglycerides and fatty acids had the same type of membrane disruption mechanism, and all types of antimicrobial lipids were lumped together mechanistically. Due to that kind of thinking, it was difficult to pursue innovation in the antimicrobial lipid field because there was no clear rationale at a molecular level why certain fatty acids or monoglycerides worked better than other ones. What we saw when reviewing the literature in this field was a need to develop new measurement strategies to do real-time interaction analysis, to deeply understand the molecular mechanisms of how fatty acids and monoglycerides disrupt membranes, Prof. Jackman says.
In our case, we were very interested in using a supported bilayer (SLB) platform, which is a two-dimensional conformal lipid bilayer coating that can coat certain types of surfaces such as silicon oxide surfaces, Prof. Jackman says. One benefit of these model membranes based on the two-dimensional SLB platform is that they are compatible with a wide range of surface-sensitive biosensing techniques. Using QCM-D, for example, you can track binding interactions, membrane shape changes, membrane disruption due to these membrane-compound interactions.
By having this controlled platform together with QCM-D monitoring, it becomes possible to test different compounds, environmental conditions such as solution pH, and varying membrane properties such as composition, or more specifically, aspects like cholesterol fraction in the bilayer.
When we think about the most powerful way to use QCM-D technology for these kinds of studies, we view it as a multi-parameter approach, Prof. Jackman says. We first measured the critical micelle concentration (CMC) of different antimicrobial lipids based on fluorescence spectroscopy, and then we defined different concentrations of antimicrobial lipid to test above and below CMC. We mainly focused on the interactions of the different compounds at different concentrations with that SLB platform by using QCM-D to study the potency of the compounds. So, identifying which concentration worked, the lowest concentration that was effective, over the concentration-dependent membrane interactions. Then we proceeded in selected cases to confirm the morphological changes with fluorescence microscopy.
When we started this work, we focused on lauric acid (LA) and glycerol monolaurate (GML), to understand how they interact with an SLB platform. We used QCM-D to collect a lot of information about the system, including the dynamics, how it changes over time, and, also in understanding what are the morphological properties of these systems in terms of their adlayer mass and viscoelasticity, Prof. Jackman says.
One thing to really emphasize here for the QCM-D is that it is highly surface-sensitive, Prof. Jackman says. The probing volume, or penetration depth, is ~100-200 nm, depending on the specific overtone. But it is very specific to things that occur at the solid-liquid interface. In our case, where we have an SLB attached to this solid liquid interface, what we are really studying with QCM-D technology is how the SLB properties change over time due to the fatty acid and monoglyceride interactions.
Based on QCM-D technology, we can study fatty acid- and monoglyceride-membrane interactions. And, before even going there, we can validate the successful fabrication of the SLB platform onto the sensor surface, also using QCM-D technology. In our case, we fabricated SLBs on silicon oxide surfaces and we ended up confirming resonance frequency shifts around -26 Hz and energy dissipation shifts of less than 0.5 ∙ 10-6. These values are consistent with supported lipid bilayers of good quality. We have demonstrated this for various kinds of fabrication techniques, and on various surfaces with QCM-D providing a very reliable tool to study this SLB platform fabrication and quality control.
After we fabricated our SLB platforms, we decided to add either lauric acid, the fatty acid, or GML monoglycerides. Comparing LA and GML, we saw extremely different membrane responses, Prof. Jackman says. In the case of the QCM-D monitoring of LA addition, we saw a very small change in the resonance frequency going down to about -40 Hz before climbing back up to around -28 Hz. And we saw a corresponding change in the energy dissipation, reaching a maximum of around 7∙10-6. When we did complementary fluorescence microscopy experiments, we saw that this LA interaction caused what we classify as tubule formation. Tubules formed that protruded from the SLB platform.
In very stark contrast, when GML was added, we saw huge changes in the resonance frequency down to about -125 Hz, along with a massive change in energy dissipation up to around over 65∙10-6 or so. This corresponded with bud formation.
So, despite previous evidence from biological studies, suggesting that fatty acids and monoglycerides have the same mechanism of membrane disruption, these biophysical studies with the QCM-D technology provided the first evidence that fatty acids and monoglycerides have distinct membrane interactions, Prof. Jackman says.
These studies also allowed us, by performing concentration-dependent experiments within the study, to confirm that micelle formation is required for extensive membrane-disruptive interactions, Prof. Jackman says. We were also able to further understand how the kind of a negative charge of LA versus the nonionic character of GML also affects rates of membrane translocation, and how this influences anisotropic vs isotropic spontaneous curvature in the SLB platform, which gives rise to the different types of membrane morphological changes. We were particularly excited that these biophysical insights gained from QCM-D measurements were recently validated by other groups using molecular dynamics solutions, validating that QCM-D is able to study molecular-level interaction phenomena.
So, based on QCM-D technology, we were able to identify for the first time that fatty acid, such as LA, induced tubulation, and monoglycerides, such as GML, induced budding. Since then, we have been able to develop cholesterol- rich SLBs, with varying levels of cholesterol, to look at how GML budding and LA tubulation is affected by the different cholesterol fractions, Prof. Jackman says.
We have also been able to develop mixtures of fatty acids and monoglycerides that have improved membrane-disruptive performance compared to either fatty acids or monoglycerides alone, Prof. Jackman says. When we mix GML and LA, we can control the CMC properties and composition of the micelles based on mixing different fractions of GML and LA together. Using QCM-D testing, we have been able to see that, whereas GML by itself causes bud formation and LA by itself causes tubule formation, when we combine GML and LA in mixed micelles, we see a biophysical competition between budding and tubule formation and that can lead to interesting biophysical phenomena. We have concluded that the LA - GML ratio is more important than the total LA - GML concentration when looking at this kind of competitive behavior between different types of possible membrane morphological responses and how they give rise to membrane disruption.
Just to conclude, QCM-D technology has been really useful for characterizing membrane interaction profiles of different antimicrobial lipids. Some of the key aspects being the ability to quantitatively characterize the potency and mechanism of action, which has been useful for performance evaluation and supporting development processes. The technology has excellent potential for medical biotechnology and there is huge opportunity for QCM-D technology in the biophysics and biotechnology space, Prof. Jackman concludes.
To learn more about how QCM-D is used the field of membrane biophysics, watch the webinar by Prof. Jackman.
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.
