This work was supported by the NSF (DMR-1710491, OISE-1743748). J.R. and E.S. acknowledge support from the NSF (DMR-1751308).

QCM-D Collagen Adsorption
1. Sample Preparation and Sensor Pre-cleaning
<ol>
	<li>Prepare 20 mL of 0.1 M acetate buffer, adjusting the pH with HCl and NaOH as necessary to achieve pH = 5.6.</li>
	<li>Add rat tail collagen solution to the 20 mL of acetate buffer under sterile conditions to a final concentration of 10 &#181;g/mL.</li>
	<li>Clean the gold-coated quartz sensor to remove organic and biological material25,26.
	<ol>
		<li>Place the sensor active side up in a UV/Ozone chamber and treat the surface for approximately 10 min.</li>
		<li>Heat a 5:1:1 mixture of deionized water (dH2O), ammonia (25%) and hydrogen peroxide (30%) to 75 &#176;C. Place the sensor in the solution for 5 min.</li>
		<li>Rinse the sensor with dH2O and dry with a stream of nitrogen gas.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. QCM-D Measurement Data Acquisition
<ol>
	<li>Turn on all necessary equipment to take a measurement including the pump, electronics unit, and computer software.</li>
	<li>Remove the flow module from the chamber platform and unscrew the large thumb screws to open the module.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using the appropriate computer software, set the temperature of the flow module to 37 &#176;C. Monitor the changing temperature for 10-15 min to ensure that it equilibrates at the desired value.</li>
	<li>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.</li>
	<li>Place the inlet pump tubing in the 1x phosphate-buffered saline (PBS) solution. Start the external pump flow at 25 &#181;L/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 &#181;L/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.</li>
	<li>Allow fluid flow of the 1x PBS through the flow module for at least 15 min to properly equilibrate.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Allow the new frequency and dissipation values to equilibrate to a stable value. Here, we expect this stabilization to occur after 8-12 h.</li>
	<li>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.</li>
	<li>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.</li>
	<li>End the data acquisition of the measurement and save the data.</li>
	<li>Clean and dismantle the QCM equipment.
	<ol>
		<li>Increase the fluid flow rate of the external pump to 500 &#181;L/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.</li>
		<li>Stop the pump and move the inlet tubing to dH2O, and resume fluid flow to further flush the system for at least 20 min.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare a 0.5 M solution of potassium bromide (KBr).
	<ol>
		<li>Dissolve 1.79 g of KBr in 30 mL of distilled water. Shake until dissolved.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare the sensor for spin coating.
	<ol>
		<li>Set the spin coat parameters to 10,000 rpm, 8,000 acceleration, and 5 s.</li>
		<li>Insert the sensor onto the spin coater and turn on the vacuum.</li>
		<li>Cover the surface of the sensor with ethanol and run the spin coater to clean the sensor surface.</li>
		<li>Add the PEC (PSS:PDADMA prepared in the same way as detailed in Sadman et al.22) to the surface of the sensor.
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Measurement of the Film in Air and Water
<ol>
	<li>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.</li>
	<li>Take a measurement of the film in air. Reference to the bare sensor in air. Allow the film data to equilibrate.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 (&#60;100 Hz).</li>
</ol>
5. Data Analysis
<ol>
	<li>Open the QCM-D data analysis MATLAB GUI created by Sadman (<a href="https://github.com/sadmankazi/QCM-D-Analysis-GUI" target="_blank">https://github.com/sadmankazi/QCM-D-Analysis-GUI</a>)27. Open the film in air data file by selecting &#34;Load QCM.&#34; 
	NOTE: The Shull group has developed a similar Python GUI for data collection and analysis for QCM (<a href="https://github.com/shullgroup/rheoQCM" target="_blank">https://github.com/shullgroup/rheoQCM</a>). A portion of the analysis code is provided in the supplementary information for both analyzing the data and generating the figures in this paper.</li>
	<li>Select the desired calculation (either 3,5,3 or 3,5,5), gamma, and film in air icons. Click Plot QCM.</li>
	<li>Determine the thickness of the dry film using the most equilibrated data point (typically the last data point) from the experiment. Record this value.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Determine the percent swelling as 
	<img alt="Equation 3" src="/files/ftp_upload/60584/60584eq3.jpg" /> (3) 
	where dp is the film thickness from the solution and dpdry is the dry film thickness.</li>
</ol>

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The authors have nothing to disclose.

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 &#62; 2 h), indicating an ability to determine viscoelastic properties of the adsorbed film.
<p cl...

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 techniques5,6.
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 mass7,8,9. 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 vessels12,13. 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 (&#915;) 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 &#915; through equation 1,14
<img alt="Equation 1" src="/files/ftp_upload/60584/60584eq1.jpg" /> (1)
where D is the dissipation factor and &#402; is the frequency. Both D and &#915; 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&#8230;). 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 group15,16,17,18.
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 experiment19,20. 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 (&#916;&#402;) 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 &#915;) 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 &#916;&#402;. However, in this region, the viscoelastic properties are reliably determined using the combination of &#916;&#402; and &#916;&#915; 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 &#916;&#402; 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 &#934; = arctan(G&#34; / G&#39;). When films are prepared in the Sauerbrey limit, the mass per unit area can be directly calculated based on the Sauerbrey equation shown below21,
<img alt="Equation 2" src="/files/ftp_upload/60584/60584eq2.jpg" /> (2)
where &#916;&#402;n is the change in the resonant frequency, n is the overtone of interest, &#402;1 is the resonant frequency of the sensor, &#916;m / A is the mass per area of the film, and Zq is the acoustic impedance of quartz, which for AT cut quartz is Zq = 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 / &#955;n parameter, which is the ratio of the film thickness (d) to the shear wavelength of the mechanical oscillation of the quartz crystal sensor (&#955;n)15,16,18. The ideal viscoelastic regime is d / &#955;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 / &#955;n is provided elsewhere15,18, 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 &#915; 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&#39;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.

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

sample preparation in quartz crystal microbalance measurements of protein adsorption and polymer mechanics

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.

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...

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

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.

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...

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Chemistry

13.2K Views.  Northwestern University. 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.

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

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

Bioengineering

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Biochemistry

Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low friction at the interface. Nevertheless, these systems can be sensitive to wear due to interdigitation of the opposing brushes. In a recent publication, we have shown via molecular dynamics simulations and atomic force microscopy experiments, that using an immiscible polymer brush system terminating the substrate and the slider surfaces, respectively, can eliminate such interdigitation. As a consequence, wear in the contacts is reduced. Moreover, the friction force is two orders of magnitude lower compared to traditional miscible polymer brush systems. This newly proposed system therefore holds great potential for application in industry. Here, the methodology to construct an immiscible polymer brush system of two different brushes each solvated by their own preferred solvent is presented. The procedure how to graft poly(N-isopropylacrylamide) (PNIPAM) from a flat surface and poly(methyl methacrylate) (PMMA) from an atomic force microscopy (AFM) colloidal probe is described. PNIPAM is solvated in water and PMMA in acetophenone. Via friction force AFM measurements, it is shown that the friction for this system is indeed reduced by two orders of magnitude compared to the miscible system of PMMA on PMMA solvated in acetophenone.

The methodology to perform friction force microscopy experiments for contacting brushes is presented: Two polymer brushes that are grafted from (a) substrates and (b) colloidal probes are slid to show that, by using two contacting immiscible brush systems, friction in sliding contacts is reduced compared to miscible brush systems.

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low ...

Engineering

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction

Source: Matos, A. L. L., et al. <a href="https://app.jove.com/v/58224">Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator.</a> J. Vis. Exp. (2018).
This video demonstrates a dissipative microgravimetry technique to investigate the binding of proteins to lipid bilayers. Small unilamellar vesicles are added to coat the surface of the quartz sensor of a microbalance. Utilizing the calcium ion-dependent binding of the target phospholipid-binding protein to the bilayer, changes in the oscillation frequency of the sensor and the dissipation of the oscillation are monitored to determine the nature of the protein-lipid bilayer binding.

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

Summary

Explore More Videos

Chapters in this video

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

Monitoring Protein Adsorption with Solid-state Nanopores

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction