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

Podsumowanie

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

Streszczenie

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

Wprowadzenie

The quartz crystal microbalance (QCM) leverages the piezoelectric effect of a quartz crystal to monitor its resonant frequency, which is dependent on the mass adhered to the surface. The technique compares the resonant frequency and bandwidth of an AT cut quartz crystal sensor (typically in the range of 5 MHz)¹ in air or a fluid to the frequency and bandwidth of the sensor after deposition of a film. There are several benefits for using the QCM to study thin film properties and interfaces, including the high sensitivity to mass and potentially to viscoelastic property changes (depending on sample uniformity and thickness), the ability to perform studies in situ², and the ability to probe a much shorter rheological timescale than traditional shear rheology or dynamic mechanical analysis (DMA). Probing a short rheological timescale allows observation of how the response at this timescale changes both over extremely short (ms)³ and long (years) durations⁴. This capability is beneficial for the study of a variety of kinetic processes and is also a useful extension of traditional rheometric techniques^5,6.

The high sensitivity of the QCM has also led to its heavy use in biological applications studying the fundamental interactions of extremely small biomolecules. An uncoated or functionalized sensor surface can be used to investigate protein adsorption; even further, biosensing through complex binding events between enzymes, antibodies, and aptamers can be examined based on changes in mass^7,8,9. For instance, the technique has been used to understand the transformation of vesicles to a planar lipid bilayer as a two-phase process of adsorption of fluid-containing vesicles to a rigid structure by observing correlating changes in frequency and viscoelasticity¹⁰. In recent years, the QCM has additionally offered a robust platform to monitor drug delivery by vesicles or nanoparticles¹¹. At the intersection of materials engineering and molecular and cellular biology, we can use the QCM to elucidate key interactions between materials and bioactive components like proteins, nucleic acids, liposomes, and cells. For example, protein adsorption to a biomaterial mediates downstream cellular responses such as inflammation and is often used as a positive indicator of biocompatibility, while in other instances extracellular protein attachment to coatings that interface with blood could induce dangerous clotting in vessels^12,13. The QCM can therefore be used as a tool to select candidates optimal for different needs.

Two common approaches for performing QCM experiments collect analogous data from the experiment: the first approach records the frequency shift and the half bandwidth (Γ) of the conductance peak. The second approach, QCM with dissipation (QCM-D), records the frequency shift and the dissipation factor, which is directly proportional to Γ through equation 1,¹⁴

(1)

where D is the dissipation factor and ƒ is the frequency. Both D and Γ are related to the damping effect the film has on the sensor, which gives an indication of the stiffness of the film. The subscript n denotes the frequency overtone or harmonic, which are the odd resonant frequencies of the quartz sensor (n = 1, 3, 5, 7…). Further discussion of models using multiple harmonics to obtain the mass and viscoelastic properties of a film can be found in a review by Johannsmann¹⁴ and previous papers from the Shull group^15,16,17,18.

One key consideration for preparing QCM samples is how to apply the thin film on the sensor surface. Some common methods include spin coating, dip coating, drop coating, or adsorption of the film onto the sensor surface during the experiment^19,20. There are four regions for QCM samples: the Sauerbrey limit, the viscoelastic regime, the bulk regime, and the overdamped regime. For sufficiently thin films, the Sauerbrey limit applies, where the frequency shift (Δƒ) provides the surface mass density of the film. Within the Sauerbrey limit, the frequency shift scales linearly with the resonant harmonic, n, and changes in damping factor (D or Γ) are generally small. In this regime sufficient information is not available to uniquely determine the rheological properties of the layer without making additional assumptions. Data in this regime are used to calculate the surface mass density (or thickness if the density is known a priori) of the film. In the bulk regime where the medium in contact with the crystal is sufficiently thick, the evanescent shear wave propagates into the medium before being completely dampened. Here, no mass information can be obtained using Δƒ. However, in this region, the viscoelastic properties are reliably determined using the combination of Δƒ and ΔΓ ^15,18. In the bulk regime, if the medium is too rigid, the film will damp out the resonance of the sensor, preventing the collection of any reliable data from the QCM. The viscoelastic regime is the intermediate regime where the film is thin enough to have the shear wave fully propagate through the film as well as have reliable values for the damping factor. The damping factor and Δƒ can then be used to determine the viscoelastic properties of the film as well as its mass. Here, the viscoelastic properties are given by the product of the density and the magnitude of the complex shear modulus |G*|p and the phase angle given by Φ = arctan(G" / G'). When films are prepared in the Sauerbrey limit, the mass per unit area can be directly calculated based on the Sauerbrey equation shown below²¹,

(2)

where Δƒ_n is the change in the resonant frequency, n is the overtone of interest, ƒ₁ is the resonant frequency of the sensor, Δm / A is the mass per area of the film, and Z_q is the acoustic impedance of quartz, which for AT cut quartz is Z_q = 8.84 x 10⁶ kg / m²s. The viscoelastic regime is most appropriate for the study of polymer films, and the bulk limit is useful for studying viscous polymer²² or protein solutions¹⁶. The different regimes depend on the properties of the material of interest, with the optimum thickness for full viscoelastic and mass characterization generally increasing with the film stiffness. Figure 1 describes the four regions with respect to the areal density of the film, complex shear modulus, and phase angle, where we have assumed a specific relationship between the phase angle and the film stiffness that has been shown to be relevant to materials of this type. Many films of practical interest are too thick for studying the viscoelastic properties with QCM, such as certain biofilms, where the thicknesses are on the order of tens to hundreds of microns²³. Such thick films are generally not appropriate for studying using the QCM, but may be measured using much lower frequency resonators (such as torsional resonators)²³, allowing the shear wave to propagate further into the film.

To determine which regime is relevant for a given QCM sample, it is important to understand the d / λ_n parameter, which is the ratio of the film thickness (d) to the shear wavelength of the mechanical oscillation of the quartz crystal sensor (λ_n)^15,16,18. The ideal viscoelastic regime is d / λ_n = 0.05 - 0.2¹⁸, where values below 0.05 are within the Sauerbrey limit and values above 0.2 approach the bulk regime. A more rigorous description of d / λ_n is provided elsewhere^15,18, but it is a quantitative parameter delineating the Sauerbrey limit and the viscoelastic limit. The analysis programs used below provide this parameter directly.

There are some additional limitations to analyzing thin films with the QCM. The Sauerbrey and viscoelastic calculations assume the film is homogeneous both throughout the film thickness and laterally across the electrode surface of the QCM. While this assumption makes it challenging to study films which have voids or fillers present, there have been some QCM investigations into films consisting of grafted nanoparticles⁶. If the heterogeneities are small compared to the overall film thickness, reliable viscoelastic properties of the composite system can still be obtained. For more heterogeneous systems, values obtained from a viscoelastic analysis should always be viewed with great caution. Ideally, results obtained from systems with unknown heterogeneity should be validated against systems which are known to be homogeneous. This is the approach we have taken in the example system described in this paper.

An important point that we illustrate in this paper is the exact correspondence between QCM measurements done in the frequency domain (where Γ is reported) and the time domain experiments (where D is reported). Results from two different QCM experiments, one time domain and one frequency domain, are described, each involving a different but conceptually related model system. The first system is a simple example of collagen attachment to the sensor to illustrate representative binding kinetics and equilibration of adsorption over time during a time domain (QCM-D) measurement. Collagen is the most abundant protein in the body, known for its versatility of binding behaviors and morphology. The collagen solution used here does not require additional functionalization of the sensor's gold surface to induce adsorption⁹. The second experimental system is a polyelectrolyte complex (PEC) composed of anionic polystyrene sulfonate (PSS) and cationic poly(diallyldimethylammonium) (PDADMA) prepared in the same fashion as Sadman et al.²². These materials swell and become soft in salt (KBr in this case) solutions, offering a simple platform for studying polymer mechanics using a frequency domain approach (QCM-Z). For each protocol, the process of preparing, taking, and analyzing a measurement is shown in Figure 2. The schematic shows that the main difference between the QCM-Z and QCM-D approaches is in the data collection step and the instrumentation used in the experiment. All the mentioned sample preparation techniques are compatible with both approaches, and each approach can analyze samples in the three regions depicted in Figure 1.

Our data demonstrate that the preparation of samples, whether by sensor coating before or during a measurement, dictates the ability to extract the viscoelastic properties of a system. By designing the early stages of an experiment appropriately, we can determine what information we can accurately gather during the analysis step.

Protokół

QCM-D Collagen Adsorption

1. Sample Preparation and Sensor Pre-cleaning

1. Prepare 20 mL of 0.1 M acetate buffer, adjusting the pH with HCl and NaOH as necessary to achieve pH = 5.6.
2. Add rat tail collagen solution to the 20 mL of acetate buffer under sterile conditions to a final concentration of 10 µg/mL.
3. Clean the gold-coated quartz sensor to remove organic and biological material^25,26.
   1. Place the sensor active side up in a UV/Ozone chamber and treat the surface for approximately 10 min.
   2. Heat a 5:1:1 mixture of deionized water (dH₂O), ammonia (25%) and hydrogen peroxide (30%) to 75 °C. Place the sensor in the solution for 5 min.
   3. Rinse the sensor with dH₂O and dry with a stream of nitrogen gas.
   4. Place the sensor active side up in a UV/Ozone chamber and treat the surface for 10 min.
      NOTE: The cleaning procedure should be immediately performed before a measurement to minimize environmental contamination on the sensor surface.

2. QCM-D Measurement Data Acquisition

1. Turn on all necessary equipment to take a measurement including the pump, electronics unit, and computer software.
2. Remove the flow module from the chamber platform and unscrew the large thumb screws to open the module.
3. If the sensor has been left out after initial cleaning (steps 1.3.1-1.3.4), rinse the sensor with deionized water (dH₂O) and dry with a stream of nitrogen gas to ensure that there are no contaminants on the surface.
4. Mount the sensor in the flow module on the exposed O-ring, first drying the area with a stream of nitrogen gas and checking that the O-ring is lying flat. The sensor should be placed with the active surface side down and anchor-shaped electrode oriented toward the marker in the flow module.
5. Turn the thumb screws to seal the flow module and replace it on the chamber platform. Attach any necessary PTFE pump tubing to the flow module and external pump.
6. Using the appropriate computer software, set the temperature of the flow module to 37 °C. Monitor the changing temperature for 10-15 min to ensure that it equilibrates at the desired value.
7. Find the initial resonance frequencies of the sensor. If any resonance frequencies are not found by the software, check that the flow module is correctly positioned on the chamber platform or re-mount the sensor in the flow module to ensure that it is centered and making proper electrical contact.
8. Place the inlet pump tubing in the 1x phosphate-buffered saline (PBS) solution. Start the external pump flow at 25 µL/min and visually inspect the tubing to be sure that the fluid is flowing through the tube.
   NOTE: Fluid flow may be easier to see by momentarily increasing the fluid flow rate to 100 µL/min or greater. If fluid does not appear to be moving through the tube, it is most likely that the two parts of the flow module are not creating a proper seal. Try tightening the thumb screws, tightening the connectors of the tubing to the inlet and outlet, or re-mounting the sensor to be sure that the O-ring is flat and centered.
9. Allow fluid flow of the 1x PBS through the flow module for at least 15 min to properly equilibrate.
10. Start the measurement in the computer software to begin data acquisition. Monitor the frequency and dissipation values for at least 5 min to ensure a stable baseline.
11. 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.
12. Allow the new frequency and dissipation values to equilibrate to a stable value. Here, we expect this stabilization to occur after 8-12 h.
13. 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.
14. Allow the new frequency and dissipation values to equilibrate to a stable value. Here, this stabilization occurs after 30 min.
    NOTE: Steps 2.13 and 2.14 can be repeated for each new period of fluid flow in more rigorous experiments with a greater number of stages.
15. End the data acquisition of the measurement and save the data.
16. Clean and dismantle the QCM equipment.
    1. Increase the fluid flow rate of the external pump to 500 µL/min or greater and place the inlet tubing into a solution of 2% Hellmanex cleaning solution for at least 20 min.
       NOTE: For other experiments, if further analysis of the sensor is desired, remove the sensor before step 2.16.1 and place another cleaning sensor in the module.
    2. Stop the pump and move the inlet tubing to dH₂O, and resume fluid flow to further flush the system for at least 20 min.
    3. Stop fluid flow and remove the sensor from the flow module. Dry the sensor and inside of the flow module with a stream of nitrogen gas. Turn off the computer software, electronics unit, and peristaltic pump.
       NOTE: The gold-coated sensors can be properly cleaned, as detailed in steps 1.3.1-1.3.4, and reused for several measurements. Indications that a sensor can no longer be reused for reliable measurements may include but are not limited to large variability in initial resonance frequencies and significant drifts in baseline measurements with buffer flow. Data can be opened and analyzed in the preferred software, including those provided by companies that specialize in QCM-D equipment.

QCM Polyelectrolyte Complex Swelling

3. Sample Preparation

NOTE: This experiment was performed using a MATLAB program developed within the Shull research group for data collection and analysis.

1. 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.
2. Submerge the sample holder in a lipless 100 mL beaker filled with distilled water and collect a reference conductance spectrum for the bare sensor in water.
3. Prepare a 0.5 M solution of potassium bromide (KBr).
   1. Dissolve 1.79 g of KBr in 30 mL of distilled water. Shake until dissolved.
   2. Insert a small silicon wafer into the KBr solution at an angle to create a slide for the quartz sensor during the annealing step to prevent the film from coming off the sensor.
4. Prepare the sensor for spin coating.
   1. Set the spin coat parameters to 10,000 rpm, 8,000 acceleration, and 5 s.
   2. Insert the sensor onto the spin coater and turn on the vacuum.
   3. Cover the surface of the sensor with ethanol and run the spin coater to clean the sensor surface.
   4. Add the PEC (PSS:PDADMA prepared in the same way as detailed in Sadman et al.²²) to the surface of the sensor.
      1. If the complex is in two phases (polymer rich and polymer poor), slowly insert the pipet into the solution. Evacuate the pipet by blowing bubbles while moving the pipet into the denser polymer rich phase.
      2. After releasing a couple bubbles in the polymer rich phase, draw up 0.5-0.75 mL of the polymer rich solution into the pipet. Maintaining pressure on the pipet bulb to not allow the polymer poor phase to enter the pipet, draw the pipet out of the solution.
      3. Wipe the outside of the pipet using a Kimwipe. Add enough solution dropwise 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.
5. Spin coat the PEC sample and immediately submerge the sensor in the 0.5 M KBr solution to prevent salt crystallization on the film.
   NOTE: This step is sometimes difficult to coordinate. Release the sensor just above the KBr solution for best results.
6. Allow the film to anneal for at least 12 h.
   NOTE: For ease of performing the experiment, prepare step 4 in the evening and allow the film to anneal overnight.

4. Measurement of the Film in Air and Water

1. Transfer the sensor to a beaker filled with distilled water to remove the excess KBr from the film and back side of the sensor. Leave the sensor in the solution for 30-60 min.
2. Take a measurement of the film in air. Reference to the bare sensor in air. Allow the film data to equilibrate.
3. Insert dried calcium sulfate into a 100 mL lipless beaker and measure the completely dry film thickness. Remove calcium sulfate from the beaker and rinse the beaker with distilled water.
4. Fill the 100 mL lipless beaker with 30 mL of distilled water. Insert a stir bar to ensure the water is circulating around the film. Measure the film in water for about 30-45 min or until the film data are equilibrated. Reference to the bare sensor in water.
5. Prepare a 15 mL solution of 3 M KBr in distilled water. Measure 5.35 g of KBr into a graduated cylinder and fill to 15 mL with distilled water. Swirl until dissolved.
6. Add the KBr solution to the beaker with distilled water in 0.1 M increments. Table 1 outlines the 0.1 M increments in mL of 3 M KBr solution. Face the film away from where the KBr solution is being added to the water so that the film does not dissolve. Make sure the system has equilibrated before adding another addition of the KBr solution.
7. 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 (30-60 min) and air dry the film.
8. To clean the PEC film from the sensor, add KBr to the beaker and gently swirl the solution. Allow to sit for 5-10 min. Repeat this process 2-3 times, then rinse the sensor with distilled water.
   NOTE: The sensor can be cleaned and reused if the response from the sensor is still good. This can be checked by the sensor having small absolute bandwidth readings for the harmonics of interest (<100 Hz).

5. Data Analysis

1. Open the QCM-D data analysis MATLAB GUI created by Sadman (https://github.com/sadmankazi/QCM-D-Analysis-GUI)²⁷. Open the film in air data file by selecting "Load QCM."
   NOTE: The Shull group has developed a similar Python GUI for data collection and analysis for QCM (https://github.com/shullgroup/rheoQCM). A portion of the analysis code is provided in the supplementary information for both analyzing the data and generating the figures in this paper.
2. Select the desired calculation (either 3,5,3 or 3,5,5), gamma, and film in air icons. Click Plot QCM.
3. Determine the thickness of the dry film using the most equilibrated data point (typically the last data point) from the experiment. Record this value.
4. Open the film in water data file. Select the same parameters as in Step 5.2, except for film in water instead of film in air.
5. After each equilibration step of the swelling experiment, determine the film thickness, complex shear modulus, and the viscoelastic phase angle. Record these values along with the ionic strength (ranging from 0-1 M in 0.1 M increments).
6. Determine the percent swelling as
   (3)
   where dp is the film thickness from the solution and dp_dry is the dry film thickness.

Wyniki

The changes in frequency with time during protein adsorption exhibit a characteristic curve and plateau shown in Figure 3A-B. The initial buffer wash of 1x PBS across the bare sensor surface induces only negligible changes in frequency, offering a steady baseline to act as a reference for future data points. The introduction of collagen solution causes protein adsorption to begin, observed as a steady decrease in frequency over time, until the density of adhered collagen pla...

Dyskusje

The collagen adsorption results span the Sauerbrey and viscoelastic regimes. By plotting the frequency shifts normalized to the corresponding harmonic number, we observe that the Sauerbrey limit holds true for approximately the first 2 h of the measurement. With increasing mass adhering to the sensor, however, the normalized frequency shifts for the third and fifth harmonics begin to deviate from one another (t > 2 h), indicating an ability to determine viscoelastic properties of the adsorbed film.

Materiały

Name	Company	Catalog Number	Comments
Acetic acid	Sigma-Aldrich	A6283	For collagen adsorption
Ammonium hydroxide solution	Sigma-Aldrich	221228	For collagen adsorption
Aqueous QCM probe	AWSensors	CLS 00050 A	For polyelectrolyte swelling
Collagen I Rat Protein, Tail	Thermo Fisher Scientific	A1048301	For collagen adsorption
Distilled water	Sigma-Aldrich	EM3234	For polyelectrolyte swelling; generally easy to acquire in research labs, but there is a catalog number in case it is not accessible
Ethanol	Sigma-Aldrich	793175-1GA-PB	For polyelectrolyte swelling
Gibco Phosphate Buffered Saline	Thermo Fisher Scientific	20012-027	For collagen adsorption
Hellmanex III	Sigma-Aldrich	Z805939	For collagen adsorption
Hydrogen peroxide solution	Sigma-Aldrich	216763	For collagen adsorption
Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply	Fisher Scientific	06-666A	For polyelectrolyte swelling
NP2K VNA	Makarov Instruments		For polyelectrolyte swelling
Poly(diallyldimethylammonium chloride), MW 200,000	Sigma-Aldrich	409022	For polyelectrolyte swelling; for full synthesis procedure see Sadman et al.
Poly(styrene-sulfonate) sodium salt 30% weight in water	Sigma-Aldrich	561967-500G	For polyelectrolyte swelling; for full synthesis procedure see Sadman et al.
Potassium Bromide	Sigma-Aldrich	793604-1KG	For polyelectrolyte swelling
QSense QCM Explorer System	Biolin Scientific		For collagen adsorption
Sodium acetate, anhydrous	Sigma-Aldrich	S2889	For collagen adsorption
Spin coater, Model WS-650MZ-23NPP	Laurell technologies		For polyelectrolyte swelling