The authors would like to acknowledge the financial support from NSF (CBET 1638893), (CBET 1638896), NIH (P20 GM113131), and the Hamel Center for Undergraduate Research at UNH. Further, the authors would like to recognize the assistance of Darcy Fournier for the assistance in cabling and Scott Greenwood for access to the DLS.

1. Example polymer preparations
<ol>
	<li>pNIPAM polymer synthesis 
	NOTE: This preparation produces 10 mL of 1 g/L solution, which is enough for 3-4 experiments.
	<ol>
		<li>Prepare the Schlenk line apparatus. Ensure the cold trap Dewar flask is filled with a slurry of dry ice and acetone, or if a mechanical refrigeration trap is used, ensure that the trap has reached an appropriate temperature.</li>
		<li>In a 50 mL round-bottom flask, add 0.566 g of N-isopropylacrylamide (NIPAM) monomer, 0.016 g of reversible addition fragmentation chain transfer polymerization (RAFT) agent (phthalimidomethyl butyl trithiocarbonate), 0.0008 g of 2,2-azobis(2-methylpropionitrile) (AIBN) and 10 mL of 1,4-dioxane. Put a stir bar into the flask. Seal the flask with a rubber septum, wrap with vinyl tape, and dissolve the monomers in 1,4-dioxane.</li>
		<li>Perform freeze-pump-thaw degassing as follows: Freeze the solution by immersing the round-bottom flask in a Dewar flask containing a slurry of dry ice and methanol. Once all material is frozen, use the vacuum manifold of the Schlenk line to evacuate the flask to an internal pressure under 100 kPa. Isolate the flask, and thaw under static vacuum, using warm water. Return the flask to atmospheric pressure using the nitrogen manifold of the Schlenk line.</li>
		<li>Repeat step 1.1.3 three times to minimize the internal oxygen concentration.</li>
		<li>Sparge the solution with nitrogen to balance the pressure to atmosphere. Heat the mixture to 85 &#176;C using an oil bath and stir at 200 rpm for 36 h.</li>
		<li>To a 50 mL beaker, add 40 mL of hexane. Then add the polymer mixture to the hexane dropwise. The pNIPAM should precipitate out as white floccule. 
		NOTE: NIPAM monomer is soluble in hexane, but pNIPAM has a poor solubility in hexane.</li>
		<li>Pour the cloudy mixture into a B&#252;chner funnel to collect the white pNIPAM powder. Transfer the powder to a 20 mL vial and put it in a vacuum oven overnight to remove leftover solvent. Store in a sealed container at room temperature until needed.</li>
	</ol>
	</li>
	<li>pNIPAM-block-poly(ferrocenylmethyl methacrylate) block-copolymer (p(NIPAM-b-FMMA)) synthesis 
	NOTE: This preparation produces 10 mL of 1 g/L solution, which is enough for 3-4 experiments.
	<ol>
		<li>Prepare the Schlenk line apparatus. Ensure the cold trap Dewar flask is filled with a slurry of dry ice and acetone, or if a mechanical refrigeration trap is used, ensure that the trap has reached an appropriate temperature.</li>
		<li>In a 50 mL round-bottom flask, add 0.057g of ferrocenylmethyl methacrylate (FMMA) monomer, 0.016 g of RAFT agent, 0.0008 g of AIBN and 10 mL of 1,4-dioxane. Put a stir bar into the flask. Seal the flask with a rubber septum, wrap with vinyl tape, and dissolve the monomers in 1,4-dioxane.</li>
		<li>Perform freeze-pump-thaw degassing as follows: Freeze the solution by immersing the round-bottom flask in a Dewar flask containing a slurry of dry ice and methanol. Once all material is frozen, use the vacuum manifold of the Schlenk line to evacuate the flask to an internal pressure under 100 kPa. Isolate the flask, and thaw under static vacuum, using warm water. Return the flask to atmospheric pressure using the nitrogen manifold of the Schlenk line.</li>
		<li>Repeat step 1.2.3 three times to minimize the internal oxygen concentration.</li>
		<li>Sparge the solution with nitrogen to balance the pressure to atmosphere. Heat the mixture to 85 &#176;C using an oil bath and stir it for 10 h.</li>
		<li>Dissolve 0.543 g of NIPAM and 0.0002 g of AIBN into 3 mL of 1,4-dioxane. Add the solution into the flask under nitrogen and sparge for 30 min. Heat the mixture to 85 &#176;C using an oil bath and stir it at 200 rpm for another 36 h.</li>
		<li>Add 40 mL of hexane to a 50 mL beaker. Then add the polymer mixture to the hexane dropwise. The p(NIPAM-b-FMMA) should precipitate out as brown powder because the FMMA monomer has a dark yellow color. 
		NOTE: NIPAM and FMMA monomers are soluble in hexane, but p(NIPAM-b-FMMA) has poor solubility in hexane.</li>
		<li>Pour the yellow cloudy mixture into a B&#252;chner funnel to collect the brown p(NIPAM-b-FMMA) powder. Transfer the powder to a 20 mL vial and put it in vacuum oven overnight to remove leftover solvent. Store in a sealed container at room temperature until needed.</li>
	</ol>
	</li>
</ol>
2. DLS sample and cuvette preparation
NOTE: This section prepares the cuvette for applied voltage and the sample for DLS measurements.
<ol>
	<li>Measure 10 mg of polymer powder and dissolve in 10 mL of filtered deionized (DI) water. Put the mixture in a refrigerator overnight. When ready to start the experiment, keep the sample on ice. 
	NOTE: The polymer concentration used in these experiments was 1 g/L, but the optimal range of concentrations for each sample will be unique. Also, best practice is to keep the polymer below the LCST until it is ready for testing.</li>
	<li>Cut two pieces of 6.3 mm x 7 cm single-sided copper tape (Figure 1). Use tweezers to stick each piece to opposite sides of the inside of the DLS sample cuvette, perpendicular to the light path. The bottom of the tape should reach near the bottom of the cuvette. Fold the edges of the copper tape over the top of the cuvette. Make sure the copper tape is near/wrapped on the top of the sample cuvette to ensure good electrical contact. Also make sure the copper tape does not connect with the metal contacts associated with the DLS equipment used for Zeta potential measurements.</li>
	<li>Wash the cuvette with DI water three times, then dab the excess water off with a Kimwipe.</li>
</ol>
3. DLS instrument controls and set up
NOTE: Three controls are recommended to complete before running each DLS experiment: (1) blank water solution; (2) a size standard; (3) measurement of the polymer before the start of a temperature ramp or applied voltage. Please consult the instrument manual before operation for guidance on preparing a sample, choosing settings, and assessing sample and data quality.
<ol>
	<li>Transfer 1.5 mL of filtered solvent to the cuvette. Use DI water.</li>
	<li>Insert the cuvette to the cuvette holder, ensuring the small arrow on the top of the cuvette is aligned with the cuvette holder. Close the lid.</li>
	<li>Within the Zetasizer software, select Measure on the toolbar. Manual measurements were set up for the controls. Set the Temperature to the experimental starting point. Select 20 &#176;C for this experiment.</li>
	<li>Once the text at the bottom of the window says, Insert cell and press start when ready, hit the green triangle start button at the top of the screen. This starts the experiment, and the cuvette holder should not be opened after this.</li>
	<li>Click on the tab Multi-view to observe real-time results. Continuously monitor the sample and data quality by observing count rate and the correlation function. Because this sample is just solvent, no clear signal corresponding to the presence of particles should be observed.</li>
	<li>Add two drops of a standard solution to the cuvette or just use the water control, and repeat steps 3.2-3.6. Use a 20 nm NIST-traceable polystyrene size standard for this experiment. 
	NOTE: If the water or standard solution control runs show data that are inconsistent with the expected results, troubleshoot the error and repeat until the controls read as expected.</li>
	<li>Rinse the cuvette and add the filtered polymer/test solution. Repeat steps 3.2-3.5. A clear measurement of the initial test solution should be observed. It is recommended to do this before any temperature ramp or applied voltage for a baseline measurement.</li>
</ol>
4. DLS SOP set up
NOTE: This section refers specifically to the temperature ramping operation of a Malvern Zetasizer NanoZS DLS instrument. Before starting experiments, it is strongly recommended to consult the instrument manual extensively for guidance on selecting a cell, preparing a sample, choosing measurement settings, and assessing sample and data quality.
<ol>
	<li>Within the Zetasizer software (version 7.11), choose File, then click New to set up a new SOP (Figure 2).</li>
	<li>Click Measurement type to select Trend &#62; Temperature &#62; Size.</li>
	<li>In Material, choose the appropriate material and refractive index. Choose Protein and the refractive index (RI) of 1.450 for this experiment. If exact values for refractive index are desired for more accurate calculation of volume distribution, the experimenter should determine the refractive index of their sample experimentally.</li>
	<li>In Dispersant, choose the appropriate solvent. Choose Water as a solvent in this experiment.</li>
	<li>In Cell, choose the cuvette being used. Use Disposable cuvettes (DTS0012) for this experiment.</li>
	<li>In Sequence, set Start temperature and End temperature. For heating experiments, set the Start temperature at 20 &#176;C and set the End temperature as 40 &#176;C. For cooling experiments, choose the opposite. Uncheck the Return to starting temperature box.</li>
	<li>Select an interval for each temperature step change. For these experiments, select 1.5 &#176;C.</li>
	<li>In Size measurement, set Equilibration time. For these experiments, set the duration to 120 s. Choose number of measurements. Choose 3 measurements and Automatic for measurement duration.</li>
	<li>Save SOP, then close the file.</li>
	<li>If applied voltage is to be used, set up the potentiostat (Section 5) before continuing.</li>
	<li>Once the potentiostat is set up, or if applied voltage is not used, return to the Zetasizer software and click Measure on the toolbar, then click Start SOP.</li>
	<li>Once the text at the bottom of the SOP window says, Insert cell and press start when ready, hit the green triangle start button at the top of the screen. This starts the experiment, and the cuvette holder should not be opened after this.</li>
	<li>Click on the tab Multi-view to observe real-time results. Continuously monitor the sample and data quality by observing count rate and the correlation function. See Figures 3-5 for representative experimental results.</li>
</ol>
5. Potentiostat Setup
NOTE: It is recommended to use the same computer for particle size and applied voltage operations to time-sync the data and thereby making it easier to evaluate later. Please consult the applied voltage instrument manuals for guidance on wiring set up, software consultation, and choosing appropriate parameters. A Gamry potentiostat was used in these experiments.
<ol>
	<li>Prepare two wires that are thin enough to fit through the small crevice on the upper right edge of the DLS cuvette holder area (Figure 6). On one end of the prepared wire, strip off the insulation to allow for a connection to the potentiostat. On the opposite end, solder a short alligator clamp to the wire and connect to the cuvette. Make sure the DLS sample lid is closed.</li>
	<li>Clamp the white reference potentiostat lead and the red counter potentiostat lead together to one of the prepared wires. Clamp the green working potentiostat lead and the blue working sense potentiostat lead to the other prepared wire. For this experiment, do not use the orange counter sense and black ground potentiostat leads and leave them floating. To ensure the circuit does not short, these wires should not touch any other lead or conductive surface. 
	NOTE: It does not matter which side each lead is connected to.</li>
	<li>Within the software toolbar, click Experiment, then click option E Physical Electrochemistry, and select Chronoamperometry. For the purposes of this protocol, use a simple applied voltage by applying a single voltage with current response measured over time (i.e., chronoamperometry). Regardless of the specific electrochemical methodology, it is recommended to monitor the system response over time.
	<ol>
		<li>Set Pre-step, Step 1, and Step 2 Voltage vs Reference. This will be the applied voltage across the entire field/cuvette. Set Voltage to 1 V vs Reference for all three steps.</li>
		<li>Set Pre-step Delay Time. For these experiments, set to 0.5 s to make sure the system is stable at the desired voltage before recording a signal.</li>
		<li>Set the time for both Step 1 Time and Step 2 Time. This controls how long the voltage will be applied. Set both to 14,400 s to make sure the applied voltage will continue throughout the DLS experiment.</li>
		<li>Set Sample Period. This is how frequently the graph will read and record current and voltage values. Use 10.0 s in this experiment. 
		NOTE: The other settings are not significant for the data presented here. The default values in the system were used.</li>
	</ol>
	</li>
	<li>Click OK. Top toolbar will display an Active sign, indicating voltage is being applied. The current should give a moderate response (&#181;A), and not overload the potentiostat. If no signal or excessive signal is observed, the system might be connected incorrectly, and therefore, troubleshoot the error and repeat until the expected current is observed.</li>
	<li>Return to step 4.10 to start the DLS SOP.</li>
</ol>
6. Data analysis
NOTE: This section details preliminary analysis to understand the data obtained.
<ol>
	<li>Import data into preferred data analysis and graphing software.</li>
	<li>For each run within a set of measurements at a given temperature, determine the particle volume size of the peak with the largest volume percent.</li>
	<li>Calculate the average and standard deviation of the volume size over the three recorded measurements at a given temperature.</li>
	<li>For each experiment, plot average size &#177; standard deviation on the y-axis (log scale) versus temperature on the x-axis (linear scale).</li>
	<li>Import Gamry current data for analysis. Plot current data with time on the x-axis and current (in microamps) on the y-axis.</li>
	<li>In order to relate current data to particle size data, compare the timestamp of the Zetasizer data to the Gamry current timestamp. This is possible if the two types of data are collected from the same computer. Otherwise, match recorded times as best as possible.</li>
</ol>

<ol>
	<li>Xu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particuology+Light+scattering+:+A+review+of+particle+characterization+applications.">Particuology Light scattering : A review of particle characterization applications.</a> Particuology. 18, 11-21 (2015).</li><li>Szczubia&#322;ka, K., Nowakowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+micelles+formed+by+smart+terpolymers+to+stimuli+studied+by+dynamic+light+scattering.">Response of micelles formed by smart terpolymers to stimuli studied by dynamic light scattering.</a> Polymer. 44 (18), 5269-5274 (2003).</li><li>Kotsuchibashi, Y., Ebara, M., Aoyagi, T., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Dual+Temperature+Responsive+Block+Copolymers+and+Their+Potential+as+Biomedical+Applications.">Recent Advances in Dual Temperature Responsive Block Copolymers and Their Potential as Biomedical Applications.</a> Polymers. 8, 380 (2016).</li><li>Lanzalaco, S., Armelin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(N-isopropylacrylamide)+and+Copolymers:+A+Review+on+Recent+Progresses+in+Biomedical+Applications.">Poly(N-isopropylacrylamide) and Copolymers: A Review on Recent Progresses in Biomedical Applications.</a> Gels. 3, 36 (2017).</li><li>Lessard, D. G., Ousalem, M., Zhu, X. X., Eisenberg, A., Carreau, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+phase+transition+of+poly(N,N-diethylacrylamide)+in+water+by+rheology+and+dynamic+light+scattering.">Study of the phase transition of poly(N,N-diethylacrylamide) in water by rheology and dynamic light scattering.</a> Journal of Polymer Science Part B: Polymer Physics. 41, 1627-1637 (2003).</li><li>Garner, B. W., Cai, T., Hu, Z., Neogi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field+enhanced+photoluminescence+of+CdTe+quantum+dots+encapsulated+in+poly+(N-isopropylacrylamide)+nano-spheres.">Electric field enhanced photoluminescence of CdTe quantum dots encapsulated in poly (N-isopropylacrylamide) nano-spheres.</a> Optics express. 16, 19410-19418 (2008).</li><li>Gallei, M., Schmidt, B. V. K. J., Klein, R., Rehahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+Poly[styrene-+block+-(ferrocenylmethyl+methacrylate)]+Diblock+Copolymers+via+Living+Anionic+Polymerization.">Defined Poly[styrene- block -(ferrocenylmethyl methacrylate)] Diblock Copolymers via Living Anionic Polymerization.</a> Macromolecular Rapid Communications. 30, 1463-1469 (2009).</li><li>Grenier, C., Timberman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Affinity+Binding+by+a+Fluorescein+Templated+Copolymer+Combining+Covalent,+Hydrophobic,+and+Acid-Base+Noncovalent+Crosslinks.">High Affinity Binding by a Fluorescein Templated Copolymer Combining Covalent, Hydrophobic, and Acid-Base Noncovalent Crosslinks.</a> Sensors. 18, 1330 (2018).</li><li>Chiefari, J., Chong, Y. K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+Free-Radical+Polymerization+by+Reversible+Addition&#8722;Fragmentation+Chain+Transfer:+The+RAFT+Process.">Living Free-Radical Polymerization by Reversible Addition&#8722;Fragmentation Chain Transfer: The RAFT Process.</a> Macromolecules. 31, 5559-5562 (1998).</li><li>Perrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50th+Anniversary+Perspective+:+RAFT+Polymerization-A+User+Guide.">50th Anniversary Perspective : RAFT Polymerization-A User Guide.</a> Macromolecules. 50, 7433-7447 (2017).</li></ol>

The authors declare no conflicts of interest.

Applying voltage to either pNIPAM or p(NIPAM-b-FMMA) solutions changed the polymer aggregation behavior in response to temperature. With both materials, when an applied voltage was present, the polymers&#39; volume size remained high even when the solutions were cooled below their LCST. This was an unexpected result, as the trials with no voltage showed the polymers returning to their original sizes. These experiments allow us to conclude that for our temperature range, and with an applied voltage, polymer aggre...

Dynamic light scattering (DLS) is a technique to determine particle size through the use of random changes in intensity of light scattered through solution1. DLS is capable of measuring aggregation of polymers by determining particle size. For this experiment, DLS was coupled with controlled temperature changes to observe when a polymer aggregates which is indicative of exceeding the lower critical solution temperature (LCST)2,3. Below the LCST, there exists one homogeneous liquid phase; above the LCST, the polymer becomes less soluble, aggregates, and condenses out of solution. An applied voltage (i.e., applied potential or electric field) was introduced across the scattering field to observe the effects of the electric field on aggregation behavior and LCST. The application of voltage in particle sizing measurements allows for new insights into particle behavior and subsequent applications in the fields of sensors, energy storage, drug delivery systems, soft robotics, and others.
In this protocol, two example polymers were used. Poly(N-isopropylacrylamide), or pNIPAM, is a thermal sensitive polymer, which contains both a hydrophilic amide group and a hydrophobic isopropyl group on the macromolecular chain4,5. Thermal-responsive polymer materials like pNIPAM have been widely used in controlled drug release, biochemical separation, and chemical sensors in recent years3,4. The LCST literature value of pNIPAM is around 30-35 &#176;C4. pNIPAM is typically not electrochemically active. Therefore, as a second sample polymer an electrochemically-active block was added to the polymer. Specifically, ferrocenylmethyl methacrylate was used to create a poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate) block-copolymer, or p(NIPAM-b-FMMA)6,7. Both example polymers were synthesized by reversible addition fragmentation chain-transfer polymerization with controlled chain length8,9,10. The non-electrochemically active polymer, pNIPAM, was synthesized as 100 mer pure pNIPAM. The electrochemically active polymer, p(NIPAM-b-FMMA), was also 100 mer chain length, which contains 4% ferrocenylmethyl methacrylate (FMMA) and 96% NIPAM.
In this article, a protocol and methodology to study the effect of applied voltage on polymer aggregation is demonstrated. This method could also be extended to other applications of DLS, such as the analysis of protein folding/unfolding, protein-protein interactions, and agglomeration of electrostatically charged particles to name a few. The sample was heated from 20 &#176;C to 40 &#176;C to identify the LCST in the absence and presence of a 1 V applied field. Then, the sample was cooled from 40 &#176;C to 20 &#176;C without disrupting the applied field to study any hysteretic or equilibrium effects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>N-Isopropylacrylamide</td>
			<td>Tokyo Chemical Industry CO., LTD</td>
			<td>I0401-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dioxane</td>
			<td>Alfa Aesar</td>
			<td>39118</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#34;-Azobis(2-methylpropionitrile)</td>
			<td>SIGMA-ALDRICH</td>
			<td>441090-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Malvern</td>
			<td>DTS0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer NanoZS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrocenylmethyl methacrylate</td>
			<td>ASTATECH</td>
			<td>FD13136-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phthalimidomethyl butyl trithiocarbonate</td>
			<td>SIGMA-ALDRICH</td>
			<td>777072-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Gamry</td>
			<td>Reference 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of voltage in dynamic light scattering particle size analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

The real-time file output of each run in the temperature ramp is presented as a table, as seen in Figure 3. Each record can be chosen independently to see the volume size (Figure 4) and correlation coefficient (Figure 5). Volume particle size distribution (PSD) is the most accurate data to interpret the overall distribution and LCST, but the quality of data should be assessed via correlation graph (<...

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Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

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9.8K Views.  University of New Hampshire. Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

Video: Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Nanosponge Tunability in Size and Crosslinking Density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

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Chapters in this video

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

Dynamic Electrochemical Measurement of Chloride Ions

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

Nanosponge Tunability in Size and Crosslinking Density

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering