Name: sample preparation in quartz crystal microbalance measurements of protein adsorption and polymer mechanics
Uploaded: 2020-01-22T12:00:00
Description: Watch this Scientific Journal Video about Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics at JoVE.com

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</su...

<ol>
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J., Yao, K., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quartz+crystal+microbalance+study+of+protein+adsorption+on+chitosan,+chitosan/poly(vinyl+pyrrolidone)+blends+and+chitosan-graft-poly(vinyl+pyrrolidone)+surfaces.">Quartz crystal microbalance study of protein adsorption on chitosan, chitosan/poly(vinyl pyrrolidone) blends and chitosan-graft-poly(vinyl pyrrolidone) surfaces.</a> Journal of Bioactive and Compatible Polymers. 22, 195-206 (2007).</li><li>Weber, N., Pesnell, A., Bolikal, D., Zeltinger, J., Kohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+fibrinogen+adsorbed+to+the+surface+of+biomaterials+used+in+blood-contacting+medical+devices.">Viscoelastic properties of fibrinogen adsorbed to the surface of biomaterials used in blood-contacting medical devices.</a> Langmuir. 23, 3298-3304 (2007).</li><li>Johannsmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic,+mechanical,+and+dielectric+measurements+on+complex+samples+with+the+quartz+crystal+microbalance.">Viscoelastic, mechanical, and dielectric measurements on complex samples with the quartz crystal microbalance.</a> Physical Chemistry Chemical Physics. 10 (31), 4516-4534 (2008).</li><li>Denolf, G. C., Sturdy, L. F., Shull, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+rheological+characterization+of+homogeneous+polymer+films+with+the+quartz+crystal+microbalance.">High-frequency rheological characterization of homogeneous polymer films with the quartz crystal microbalance.</a> Langmuir. 30 (32), 9731-9740 (2014).</li><li>Martin, E. J., Mathew, M. T., Shull, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+electrochemically+deposited+protein/metal+complexes.">Viscoelastic properties of electrochemically deposited protein/metal complexes.</a> Langmuir. 31 (13), 4008-4017 (2015).</li><li>Sturdy, L., Casadio, F., Kokkori, M., Muir, K., Shull, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quartz+crystal+rheometry:+A+quantitative+technique+for+studying+curing+and+aging+in+artists'+paints.">Quartz crystal rheometry: A quantitative technique for studying curing and aging in artists' paints.</a> Polymer Degradation and Stability. 107, 348-355 (2014).</li><li>Sadman, K., Wiener, C. G., Weiss, R. A., White, C. C., Shull, K. R., Vogt, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Rheometry+of+Thin+Soft+Materials+Using+the+Quartz+Crystal+Microbalance+with+Dissipation.">Quantitative Rheometry of Thin Soft Materials Using the Quartz Crystal Microbalance with Dissipation.</a> Analytical Chemistry. 90 (6), 4079-4088 (2018).</li><li>Wasilewski, T., Szulczy&#324;ski, B., Kamysz, W., G&#281;bicki, J., Namie&#347;nik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+three+peptide+immobilization+techniques+on+a+qcm+surface+related+to+acetaldehyde+responses+in+the+gas+phase.">Evaluation of three peptide immobilization techniques on a qcm surface related to acetaldehyde responses in the gas phase.</a> Sensors (Switzerland). 18 (11), 1-15 (2018).</li><li>Lvov, Y., Ariga, K., Kunitake, T., Ichinose, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Multicomponent+Protein+Films+by+Means+of+Electrostatic+Layer-by-Layer+Adsorption.">Assembly of Multicomponent Protein Films by Means of Electrostatic Layer-by-Layer Adsorption.</a> Journal of the American Chemical Society. 117 (22), 6117-6123 (1995).</li><li>Sauerbrey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verwendung+von+Schwingquarzen+zur+W&#228;gung+d&#252;nner+Schichten+und+zur+Mikrow&#228;gung.">Verwendung von Schwingquarzen zur W&#228;gung d&#252;nner Schichten und zur Mikrow&#228;gung.</a> Zeitschrift f&#252;r Physik. 155 (2), 206-222 (1959).</li><li>Sadman, K., Wang, Q., Chen, Y., Keshavarz, B., Jiang, Z., Shull, K. 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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.
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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...

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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 ...

The quartz crystal microbalance is useful for sensing small concentrations of another compound and can be applied to questions regarding the mechanics of soft matter and biomaterial systems. The main advantage is that the quartz crystal microbalance is able to obtain highly accurate information for small sample sizes. When extracting mechanical property information, it is necessary to work with films of an appropriate thickness.Researchers must be careful to apply the correct modeling for a film during analysis. The technique is well-suited for studying a wide range of material systems and for understanding how mechanical properties of polymeric materials respond to the environment. After turning on all necessary equipment, remove the flow module from the chamber platform and unscrew the large thumbscrews to open the module.Dry the O-ring on the flow module with a stream of nitrogen gas and check that the O-ring is lying flat. Mount the sensor on the O-ring by placing the sensor with the active surface side down and an anchor shaped electrode oriented toward the marker in the flow module. Find the initial resonance frequencies of the sensor.Place the inlet pump tubing in the 1X PBS. Start the external pump flow at 25 microliters per minute and visually inspect the tubing to be sure that the fluid is flowing through the tube. Allow the fluid flow to properly equilibrate for at least 15 minutes.In the software, press start measurement to start the measurement and begin data acquisition. Monitor the frequency and dissipation values for at least five minutes to ensure a stable baseline. Stop the pump and move the inlet tubing to the collagen acetate buffer solution and resume fluid flow.Note the time of this event for later analysis. Allow the new frequency and dissipation values to equilibrate for eight to 12 hours to a stable value, then stop the pump, move the inlet tubing back to the 1X PBS solution and resume fluid flow. Note the time of this event for later analysis.Again, allow the new frequency and dissipation values to equilibrate for 30 minutes to a stable value. End the data acquisition of the measurement, then save the data. First, position a bare quartz crystal sensor in a sample holder connected to the vector network analyzer and computer.Turn on the analyzer to apply an oscillating voltage to the sensor and collect a reference conductance spectrum for the sensor in air, then submerge the sample holder in a lipless 100 milliliter beaker filled with distilled water and collect a reference conductance spectrum for the bare sensor in water. Insert a small silicon wafer into a 0.5 molar potassium bromide solution at an angle to create a slide for the quartz sensor during the annealing step to prevent the film from coming off of the sensor. Prepare to add the PEC to the surface of the sensor.If the PEC complex is in two phases, carefully draw up approximately 0.5 milliliters of the polymer-rich phase into the pipette. Maintaining pressure on the pipette bulb to not allow the polymer-poor phase to enter the pipette, draw the pipette out of the solution. Wipe the outside of the pipette using a chem wipe.Add enough solution drop wise onto the surface of the quartz sensor to completely cover the surface. Make sure there are no visible bubbles in the solution on the sensor surface. Spin coat the PEC sample and immediately submerge the sensor in the 0.5 molar potassium bromide solution to prevent salt crystallization on the film.Allow the film to anneal for at least 12 hours. Transfer the sensor to a beaker filled with distilled water to remove the excess potassium bromide from the film and the backside of the sensor. Leave the sensor in the solution for 30 to 60 minutes.Take a measurement of the film applied to the surface of the sensor in air referenced to the bare sensor in air. Allow the film data to equilibrate. Next, insert dried calcium sulfate into a 100 milliliter lipless beaker and measure the completely dry film thickness.Remove calcium sulfate from the beaker and rinse the beaker with distilled water. Fill the 100 milliliter lipless beaker with 30 milliliters of distilled water. Insert a stir bar to ensure the water is circulating around the film.With reference to the bare sensor in water, measure the film in water for about 30 to 45 minutes or until the film data are equilibrated. Prepare a 15 milliliter solution of three molar potassium bromide by measuring 5.35 grams of potassium bromide into a graduated cylinder and fill to 15 milliliters with distilled water. Swirl until dissolved.Face the film away from where the potassium bromide solution is being added to the water so that the film does not dissolve. Add the potassium bromide solution to the beaker with distilled water in 0.1 molar increments. Make sure the system is equilibrated before adding another addition of the potassium bromide solution.After all the data have been acquired, remove the film from the holder and place in a beaker of distilled water. Allow the salt to leave the film for 30 to 60 minutes and air dry the film. In this study, the introduction of collagen solution caused protein absorption to begin which was observed as a steady decrease in frequency and increase in dissipation over time until the density of adhered collagen plateaus at a stable baseline.The frequency changes are correlated with the mass of the film and the dissipation is correlated with energy dissipation from the film. A theoretical model is needed to obtain the viscoelastic properties quantitatively. The viscoelastic analysis of collagen using a power law model shows the aerial mass, complex shear modulus, and viscoelastic phase angle respectively.The first 10 hours indicate the main adsorption stage of the collagen to the sensor surface with the period between 10 and 20 hours showing the equilibration stage before the buffer wash performed at 20 hours. Here is the plot of the viscoelastic phase angle and the complex shear modulus over the general range of samples measured using the QCM. The green line indicates the linear relationship between the two properties.This plot shows how a PEC can demonstrate a wide range of mechanical properties based on the ratio of polymer, water and salt in the system. The sample preparation directly affects the results from a QCM experiment. Understanding what data you wish to learn from a sample informs how thick the sample should be.The QCM is very useful for sensing environmental and biological processes. It can also probe the rheological behavior in the high frequency regime for characterizing the viscoelasticity of materials.

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Chemistry

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 ...

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 ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids with adsorbates. Vibrational spectra provide information on the chemical nature of adsorbed species and their structure. Thus, they are very useful for obtaining molecular level understanding of surface species. The IR spectrum of the sample itself gives some direct information about the material. General conclusions can be drawn concerning hydroxyl groups, some stable surface species and impurities. However, the spectrum of the sample is &#34;blind&#34; with respect to the presence of coordinatively unsaturated ions and gives rather poor information about the acidity of surface hydroxyls, species decisive for the adsorption and catalytic properties of the materials. Furthermore, no discrimination between bulk and surface species can be made. These problems are solved by the use of probe molecules, substances that interact specifically with the surface; the alteration of some spectral features of these molecules as a result of adsorption provides valuable information about the nature, properties, location, concentration, etc., of the surface sites.
The experimental protocol for in-situ IR studies of gas/sample interaction includes preparation of a sample pellet, activation of the material, initial spectral characterization through the analysis of the background spectra, characterization by probe molecules, and study of the interaction with a particular set of gas mixtures. In this paper we investigate a zirconium terephthalate metal organic framework, Zr6O4(OH)4(BDC)6 (BDC = benzene-1,4-dicarboxylate), namely UiO-66 (UiO refers to University of Oslo). The acid sites of the UiO-66 sample are determined by using CO and CD3CN as molecular probes. Furthermore, we have demonstrated that CO2 is adsorbed on basic sites exposed on dehydroxylated UiO-66. Introduction of water to the system produces hydroxyl groups acting as additional CO2 adsorption sites. As a result, CO2 adsorption capacity of the sample is strongly enhanced.

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

The use of FTIR spectroscopy for investigation of the surface properties of polycrystalline solids is described. Preparation of sample pellets, activation procedures, characterization with probe molecules and model studies of CO2 adsorption are discussed.

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...