QCM-D technology is a very sensitive analysis method, and to collect high-quality data, it is important to pay careful attention to the entire process of measurement preparation, setup, and execution.
To learn more about the best practice procedures, I interviewed my colleague, Jennie Ringberg, Technical product manager for QSense. Jennie described the five steps of QCM-D measurement preparation and execution. In this post I share what I learned about step two – preparation of sample liquids.
To get the most out of QCM-D your measurement in terms of data quality and reproducibility, there are several aspects to pay attention to. The measurement setup and execution could be divided into five steps:
In a previous blogpost we talked about, step 1 - sensor preparation. In this post we continue with step two and look at what to consider in the context of preparation of sample liquids.
When it comes to preparation of sample liquids, there are quite a few things to consider, Jennie says. First, if you are planning to use a new solution of any kind, you must consider any safety regulations associated with this liquid. So, please check out the MSDS sheet.
You also need to think about the chemical compatibility, and make sure that the solutions you plan to use in your experiments are compatible with all the instrument parts that it will meet, Jennie continues. Some parts of the instrument, such as the O-rings and gaskets, and the pump tubing, need to be considered when it comes to chemical compatibility. The rest of the flow path is very chemically resistant. There are high resistant O-rings, gaskets and tubing available, but you need to be aware the chemical specification and change accordingly, Jennie says.
Another aspect to consider is to never use old buffers and solutions, Jennie says. Prepare all your solutions and use them fresh. So, what is ‘old’? This depends on the solution. For example, if you are working with PBS buffer, bacteria like to grow in it. So, for that one, the shelf life might be shorter than it may be for some other solution where bacteria do not like to grow. So, make sure nothing grows in the buffer and that there are not visible particles etc., Jennie says.
Another thing that could really affect the results of your measurement, and which is important to be aware of and plan for, is the so-called bulk effect, Jennie says. This is an effect that arises when you change the properties of the bulk. So, for example if you are working with a solution, let’s say buffer A, and then you change to buffer B, which has a completely different viscosity and density, then you would see a large shift in your f and D curves. Now, if you have added your sample in buffer B, then you may interpret that shift as an indication of that something is absorbing to the surface, when in fact it is just a bulk shift. This effect needs to be considered when you design your experiment and you need to plan for how to prepare the solvents and samples, she says.
Now, there could be situations where you need to use different buffers or different solvents, Jennie continues. Then, my first suggestion would be to start with a baseline in buffer A. Add your sample, if possible, in buffer A, and then you rinse with buffer A again. And then if you need to switch, you can after this rinse step, go from A to B which will give a bulk effect, and then move on from there so that you don't go from this adsorption step directly to buffer B.
Another approach would be to do a buffer sequence, or solvent sequence, in the beginning of the measurement with just the bare surface. This way you will know what the bulk shifts are, and you will have a reference, something to relate to, Jennie explains.
Another important aspect to consider is to prevent unnecessary temperature variation during the measurement since this will have a major impact on the signal, Jennie continues. To prevent temperature variation and temperature induced artifacts in your measurement, you should equilibrate the temperature of all the solvents and samples. For example, if you are storing your buffer or solution in the fridge and use it directly from the fridge, it will probably be a lot colder than the instrument set temperature, Tset. In this situation you are introducing a temperature variation which could potentially result in huge shifts in the QCM-D signals. To avoid this, you should always let your solutions equilibrate to a temperature as close as possible to the instrument set temperature, Jennie stresses.
Also, make sure not to heat the solution in the instrument, she continues. If you heat the solution inside the instrument, you will create bubbles. And as we say, bubble mean trouble. From this perspective, it is better that the solution is slightly warmer than Tset and that you cool the solution in the instrument.
Another thing to consider is the presence of particles, Jennie says. Particles do not work well in the measurement, so if you do have particles in your solutions such as aggregates etc., that you are not interested in analyzing, you could for example filtrate or centrifuge your sample to remove them.
Another thing to consider is the stability of your solutions, Jennie says. For example, if you are working with proteins, they tend to degrade. Especially if you increase the temperature. If you have measurement results from a native protein, and then you compare the results with results from a sample where the proteins have denatured, degraded somehow, or aggregated, the results would be very different. Or, if you unintentionally have a mix of native and degraded proteins and compare these results to results where you used native proteins, the results could be very different even though you think you have the same sample.
Having the same state of the sample is important, Jennie says. You should use fresh solutions that have been treated the same way every time so that you do not start measuring and then you let a week pass and then you run your second measurement, because then it's not to consider the same sample, she says.
Talking about proteins, it might be good to know that if you are working with, for instance, proteins and macromolecules, you cannot expect them to interact with the surface, or bind, in the same way at all concentrations, Jennie says. Sometimes they behave differently depending on the sample concentration. So, sample concentration is also important to consider when planning your experiments.
So, what is a good concentration? It is hard to give a universal answer to that because it depends on the kind of sample, Jennie continues. If the concentration is too low, you may not see the signal at all. And if you are looking at the kinetics of the process, maybe it will be too slow. But on the other hand, if the concentration is too high, the surface interaction, such adsorption, might be hidden by the bulk shift. So, somewhere in between is ideal, she says.
These are the key aspects to consider when preparing the sample liquids, Jennie concludes.
Listen to the interview with Jennie to learn more about aspects to consider and pitfalls to avoid in the sensor preparation and the other four steps involved in the QCM-D measurement preparation, setup, and execution to collect high-quality data.
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.
