Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

The quartz crystal microbalance is useful for sensing small concentrations of another compound and can be applied to questions regarding the mechanics of soft matter and biomaterial systems. The main advantage is that the quartz crystal microbalance is able to obtain highly accurate information for small sample sizes. When extracting mechanical property information, it is necessary to work with films of an appropriate thickness.

Researchers must be careful to apply the correct modeling for a film during analysis. The technique is well-suited for studying a wide range of material systems and for understanding how mechanical properties of polymeric materials respond to the environment. After turning on all necessary equipment, remove the flow module from the chamber platform and unscrew the large thumbscrews to open the module.

Dry the O-ring on the flow module with a stream of nitrogen gas and check that the O-ring is lying flat. Mount the sensor on the O-ring by placing the sensor with the active surface side down and an anchor shaped electrode oriented toward the marker in the flow module. Find the initial resonance frequencies of the sensor.

Place the inlet pump tubing in the 1X PBS. Start the external pump flow at 25 microliters per minute and visually inspect the tubing to be sure that the fluid is flowing through the tube. Allow the fluid flow to properly equilibrate for at least 15 minutes.

In the software, press start measurement to start the measurement and begin data acquisition. Monitor the frequency and dissipation values for at least five minutes to ensure a stable baseline. Stop the pump and move the inlet tubing to the collagen acetate buffer solution and resume fluid flow.

Note the time of this event for later analysis. Allow the new frequency and dissipation values to equilibrate for eight to 12 hours to a stable value, then stop the pump, move the inlet tubing back to the 1X PBS solution and resume fluid flow. Note the time of this event for later analysis.

Again, allow the new frequency and dissipation values to equilibrate for 30 minutes to a stable value. End the data acquisition of the measurement, then save the data. First, position a bare quartz crystal sensor in a sample holder connected to the vector network analyzer and computer.

Turn on the analyzer to apply an oscillating voltage to the sensor and collect a reference conductance spectrum for the sensor in air, then submerge the sample holder in a lipless 100 milliliter beaker filled with distilled water and collect a reference conductance spectrum for the bare sensor in water. Insert a small silicon wafer into a 0.5 molar potassium bromide solution at an angle to create a slide for the quartz sensor during the annealing step to prevent the film from coming off of the sensor. Prepare to add the PEC to the surface of the sensor.

If the PEC complex is in two phases, carefully draw up approximately 0.5 milliliters of the polymer-rich phase into the pipette. Maintaining pressure on the pipette bulb to not allow the polymer-poor phase to enter the pipette, draw the pipette out of the solution. Wipe the outside of the pipette using a chem wipe.

Add enough solution drop wise onto the surface of the quartz sensor to completely cover the surface. Make sure there are no visible bubbles in the solution on the sensor surface. Spin coat the PEC sample and immediately submerge the sensor in the 0.5 molar potassium bromide solution to prevent salt crystallization on the film.

Allow the film to anneal for at least 12 hours. Transfer the sensor to a beaker filled with distilled water to remove the excess potassium bromide from the film and the backside of the sensor. Leave the sensor in the solution for 30 to 60 minutes.

Take a measurement of the film applied to the surface of the sensor in air referenced to the bare sensor in air. Allow the film data to equilibrate. Next, insert dried calcium sulfate into a 100 milliliter lipless beaker and measure the completely dry film thickness.

Remove calcium sulfate from the beaker and rinse the beaker with distilled water. Fill the 100 milliliter lipless beaker with 30 milliliters of distilled water. Insert a stir bar to ensure the water is circulating around the film.

With reference to the bare sensor in water, measure the film in water for about 30 to 45 minutes or until the film data are equilibrated. Prepare a 15 milliliter solution of three molar potassium bromide by measuring 5.35 grams of potassium bromide into a graduated cylinder and fill to 15 milliliters with distilled water. Swirl until dissolved.

Face the film away from where the potassium bromide solution is being added to the water so that the film does not dissolve. Add the potassium bromide solution to the beaker with distilled water in 0.1 molar increments. Make sure the system is equilibrated before adding another addition of the potassium bromide solution.

After all the data have been acquired, remove the film from the holder and place in a beaker of distilled water. Allow the salt to leave the film for 30 to 60 minutes and air dry the film. In this study, the introduction of collagen solution caused protein absorption to begin which was observed as a steady decrease in frequency and increase in dissipation over time until the density of adhered collagen plateaus at a stable baseline.

The frequency changes are correlated with the mass of the film and the dissipation is correlated with energy dissipation from the film. A theoretical model is needed to obtain the viscoelastic properties quantitatively. The viscoelastic analysis of collagen using a power law model shows the aerial mass, complex shear modulus, and viscoelastic phase angle respectively.

The first 10 hours indicate the main adsorption stage of the collagen to the sensor surface with the period between 10 and 20 hours showing the equilibration stage before the buffer wash performed at 20 hours. Here is the plot of the viscoelastic phase angle and the complex shear modulus over the general range of samples measured using the QCM. The green line indicates the linear relationship between the two properties.

This plot shows how a PEC can demonstrate a wide range of mechanical properties based on the ratio of polymer, water and salt in the system. The sample preparation directly affects the results from a QCM experiment. Understanding what data you wish to learn from a sample informs how thick the sample should be.

The QCM is very useful for sensing environmental and biological processes. It can also probe the rheological behavior in the high frequency regime for characterizing the viscoelasticity of materials.