Why would you want to measure mass and thickness of thin films? One reason to monitor these two parameters is to characterize the build-up and degradation of molecular layers. Also, numerous materials and thin coatings are dynamic in nature and undergo physical changes responding to various stimuli, such as light, temperature, salt concentration or pH. These changes influence the mass, thickness and the structural properties of the material. The mass and thickness are therefore two parameters that are very much involved both the creation, behavior, and degradation of thin films. This makes them relevant to monitor both in the design, characterization, evaluation and optimization of thin films and coatings.
Monitoring mass and thickness changes of molecular layers
During surface interaction processes such as for example binding of molecules, adsorption, desorption, aggregation, and build-up of multilayer films, the mass and thickness of the molecular layers change. By measuring these changes in real time, we can follow the molecular binding processes and rearrangements. To measure these changes on a molecular scale, we need real-time nanoscale techniques. Once such technique is Quartz Crystal Microbalance with Dissipation monitoring technology (QCM-D). The QCM-D measures the changes of two parameters, the resonance frequency (f) and the energy dissipation (D). From these two parameters, mass and thickness changes at the surface can be extracted. In general, as the mass increases at the surface, the f will decrease. The D-parameter will indicate how soft the layer is. The softer the layer the higher the D. In the case of mass loss, the frequency will instead increase. And if the layer goes from soft to stiff, then the D parameter will decrease.
Characterizing mass uptake and mass loss using a nanogram balance
Molecules that are typically studied with this surface sensitive technology include lipids, proteins, DNA, polymers, surfactants, nanoparticles.
Let us take an example of a mass measurement. Here we are interested in detecting when an anti-biotin antibody first binds to a biotinylated lipid bilayer. We would also like to detect the cleavage of the antibody by an enzyme.
- As illustrated in Figure 1, a biotin tagged supported lipid bilayer is formed on the surface (prior to step I, and not described in this text). This is our reference surface, step I.
- We then introduce the antibody solution which is flown over the bilayer, step II. We immediately monitor a mass uptake, which means that the antibodies bind to the biotin. We let the antibodies bind until saturation, which results in a mass uptake of 1100 ng/cm2.
- Next, we introduce an enzyme, IdeS, step III. As the enzyme will cleave the anti-biotin, we are expecting a mass loss. As shown in Figure 1, this is also what we see. After the enzyme is introduced, we observe a decreased in mass density by 30%. This agrees well with the fact that 1/3 of the molecule is cut off.
This measurement shows how monitoring of the mass changes allows us to detect both the binding of the antibody and the cleavage by the enzyme. Overall, the measurement offers insight in to both the molecular interaction processes and verifies the function of the enzyme as well as the direction of the antibody at the surface.
Figure 1. Mass changes as an antibody binds to the biotinylated bilayer on the surface (I-II) followed by enzyme cleavage (III) which removes 1/3 of the mass at the surface.
How it works in practice
- Substrate preparation - The sample molecules of interest are adsorbed onto a QCM-D sensor, coated with a suitable material. Typical materials are metals, glass or plastics.
- Experimental setup – The sensor is placed in the instrument. The surface is then exposed to the samples, compounds or molecules of interest to initiate the desired adsorption/desorption phenomenon. Parameters such as temperature, humidity, osmolarity and pH can be precisely controlled during the experiment.
- Real-time monitoring - Throughout the measurement, the film mass and thickness is derived from the changes in resonance frequency (f) and energy dissipation (D) recorded by the instrument. Other film properties such as viscoelasticity and hydration can also be obtained.
Download our overview to read more about what information you can extract from QCM-D analysis.
