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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

Watch this Scientific Journal Video about Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics at JoVE.com

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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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

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

Preparation of Macroporous Epitaxial Quartz Films on Silicon by Chemical Solution Deposition

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step process based on the preparation of a sol in a one-pot synthesis which is followed by the deposition of a gel film on Si(100) substrates by evaporation induced self-assembly using the dip-coating technique and ends with a thermal treatment of the material to induce the gel crystallization and the growth of the quartz film. The formation of a silica gel is based on the reaction of a tetraethyl orthosilicate and water, catalyzed by HCl, in ethanol. However, the solution contains two additional components that are essential for preparing mesoporous epitaxial quartz films from these silica gels dip-coated on Si. Alkaline earth ions, like Sr2+ act as glass melting agents that facilitate the crystallization of silica and in combination with cetyl trimethylammonium bromide (CTAB) amphiphilic template form a phase separation responsible of the macroporosity of the films. The good matching between the quartz and silicon cell parameters is also essential in the stabilization of quartz over other SiO2 polymorphs and is at the origin of the epitaxial growth.

A protocol is presented for the preparation of piezoelectric macroporous epitaxial films of quartz on silicon by solution chemistry using dip-coating and thermal treatments in air.

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. Conductive polymers, poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-propylenedioxythiophene) (PProDOT), and a semiconducting polymer, poly(thieno[3,2-b]thiophene) (PTT), were deposited on unconventional highly-disordered and textured substrates with high surface areas, such as paper, towels and fabrics. This reported deposition chamber is an improvement of previous vapor reactors because our system can accommodate both volatile and nonvolatile monomers, such as 3,4-propylenedioxythiophene and thieno[3,2-b]thiophene. Utilization of both solid and liquid oxidants are also demonstrated. One limitation of this method is that it lacks sophisticated in situ thickness monitors. Polymer coatings made by the commonly used solution-based coating methods, such as spin-coating and surface grafting, are often not uniform or susceptible to mechanical degradation. This reported vapor phase deposition method overcomes those drawbacks and is a strong alternative to common solution-based coating methods. Notably, polymer films coated by the reported method are uniform and conformal on rough surfaces, even at a micrometer scale. This feature allows for future application of vapor deposited polymers in electronics devices on flexible and highly textured substrates.

This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper.

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...