MG, MO'C and PC thank the Molecular Medicine Research Group at WSU for Research Seed Funding, as well as Michele Mason (WSU), Richard Wuhrer (Advanced Materials Characterisation Facility, AMCF, WSU) and Herv&#233; Cottet (Montpellier) for discussions.

1. Preparation of Chitosan Films
<ol>
	<li>Weigh out 2 g of glacial acetic acid, complete to 100 ml with ultrapure water.</li>
	<li>Weigh out 1.7 g of chitosan powder, add 100 ml of the 2% m/m acetic acid aqueous solution. Stir for 5 days with stirring bar and magnetic stirring plate at room temperature either covered with aluminum foil or in the dark.</li>
	<li>Centrifuge the chitosan dispersion at 1,076 x g at 23 &#176;C for 1 hr. Collect the supernatant with a syringe and discard the precipitate.</li>
	<li>For each film, aliquot 10 ml of the chitosan suspension into a 9 cm plastic Petri dish at room temperature. Leave the films covered to dry for at least 7 days.</li>
	<li>Using scissors cut the dry films into 1 x 1 cm squares. Note: The experiment can be paused at this stage.</li>
</ol>
2. Preparation of Phosphate-buffered Saline (PBS)
<ol>
	<li>Weigh out 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate and 0.24 g potassium dihydrogen phosphate.</li>
	<li>Dissolve these weighed out chemicals in 800 ml of ultrapure water and titrate the solution with concentrated hydrochloric acid to pH 7.4. 
	Note: The experiment can be paused at this stage.</li>
</ol>
3. Preparation of 75 mM Sodium Borate Buffer at pH 9.2
<ol>
	<li>Weigh out 3.0915 g of boric acid. Dissolve it in 75 ml of ultrapure water.</li>
	<li>Titrate the boric acid solution to a pH of 9.2 with a sodium hydroxide solution at a concentration of 10 M or higher. 
	Caution: Concentrated sodium hydroxide solutions are corrosive and should be handled with gloves.</li>
	<li>Complete with ultrapure water to obtain 100 ml of solution. This yields a 500 mM sodium borate buffer at pH 9.2.</li>
	<li>Dilute the 500 mM sodium borate buffer with ultrapure water to 75 mM sodium borate buffer. Note: The experiment can be paused at this stage.</li>
</ol>
4. Preparation of Chitosan Films for the Grafting Reaction
<ol>
	<li>Rinse 10 square chitosan films (1 x 1 cm) in 5 ml of PBS for 2 hr in a Petri dish at room temperature.</li>
	<li>During this time, prepare and validate the capillary electrophoresis instrument (step 5).</li>
</ol>
5. Preparation and Validation of the Capillary Electrophoresis Instrument
<ol>
	<li>Prepare a 43.5 cm bare fused silica capillary with an internal diameter of 50 &#181;m (43.5 cm is the total length, the effective length to the detection window is typically 35 cm) by weakening the polymer outer coating of the capillary at the set length with a blunt utensil then snapping the capillary.
	<ol>
		<li>Create a window for the capillary by using a lighter to burn the polymer coating at 8.5 cm from the inlet and after it cools wipe it clean with ethanol. Burn the coating of the capillary at each end for a few millimeters with a lighter, and after it cools wipe it clean with ethanol.</li>
		<li>Place capillary inside detection window and install it in the capillary cassette by placing it at equal lengths in the inlet and outlet and winding it around the spindles of the cassette. Then install the cassette in the capillary electrophoresis instrument.</li>
	</ol>
	</li>
	<li>Set the parameters of the method for each separation. In the software menu select &#34;method&#34; then &#34;edit the entire method&#34;. Set the temperature, time, voltage, and vials used for the separation (for example 25 &#176;C, 10 min, 30 kV).
	<ol>
		<li>In the pre-conditioning section, set the consecutive flushes: 10 min with 1 M sodium hydroxide (in water), 5 min with 0.1 M sodium hydroxide (in water), 5 min with ultrapure water and 5 min with 75 mM sodium borate buffer at pH 9.2 for the first method of a series of analyses.</li>
		<li>For the subsequent methods, set the set the consecutive flushes in the pre-conditioning section: 1 min with 1 M sodium hydroxide (in water), 5 min with 75 mM sodium borate buffer at pH 9.2.</li>
		<li>In the injection section, set parameters for a hydrodynamic injection with 30 mbar pressure for 10 sec for all methods. In the separation section, set the separation conditions to 30 kV at 25 &#176;C for 9 min for all methods. 
		NOTE: Consult user manual of specific CE instrument as procedure for operating the CE instrument may vary between manufacturers. Prepare the 1 M sodium hydroxide solution on the day.</li>
	</ol>
	</li>
	<li>Inject and separate a neutral internal standard (10 &#181;l of 10% v/v dimethylsulfoxide (DMSO), in water diluted into 450 &#181;l of 75 mM sodium borate buffer). Then inject and separate in the same way an oligoacrylate standard (dissolved in ultrapure water at 10 g&#8729;L-1; see List of Materials) to check the validity of the capillary. Pause the sequence here until the grafting reaction is ready to start.</li>
</ol>
6. Grafting of RGDS onto Chitosan Film
<ol>
	<li>Weigh out the peptide (1 mg RGDS) and the coupling agents (3 mg EDC-HCl and 2 mg NHS).</li>
	<li>2 hr after the start of the chitosan film soaking in PBS, dissolve the peptide and the coupling agents in 5 ml of PBS.
	<ol>
		<li>Take a 50 &#181;l aliquot of this solution. Add 2 &#181;l of 10% v/v DMSO in water as an internal neutral standard to the aliquot. Analyze the aliquot with CE (see step 7).</li>
	</ol>
	</li>
	<li>Remove the 5 ml of PBS used to rinse the chitosan films from the Petri dish. Add the 5 ml solution of peptide and coupling agents to the Petri dish containing the chitosan films.</li>
	<li>Cover the Petri dish with paraffin film and place it on an orbital shaker at room temperature. Take 50 &#181;l aliquots of reaction media at set times. 
	NOTE: The total analysis time with CE is 15 min, thus an aliquot can be taken every 15 min (or every 30 min if two reactions are monitored in parallel, etc.).
	<ol>
		<li>Add 2 &#181;l of 10% v/v DMSO in water as an internal neutral standard to each aliquot. 
		NOTE: Aliquots should be analyzed with CE as soon as they are taken (see step 7).</li>
	</ol>
	</li>
	<li>After 4 hr of shaking and aliquot removal, remove the Petri dish from the shaker. Remove the reaction medium from the Petri dish. Add 5 ml of PBS to rinse the chitosan films.</li>
	<li>Remove the PBS from the Petri dish, rinse the chitosan film with ultrapure water and allow them to dry overnight. Remove the ultrapure water and store the films at -20 &#176;C in a plastic Petri dish.</li>
</ol>
7. Monitoring of Grafting Reaction Using CE
<ol>
	<li>Inject and separate aliquots of reaction media immediately after removal from the Petri dish using the analysis conditions as in section 5.2.</li>
	<li>Upon completion of the separations rinse the capillary with ultrapure water for 10 min. Dry it through a flush with an empty vial (air) for 10 min. 
	NOTE: The experiment can be paused at this stage.</li>
</ol>
8. Data Treatment for CE
<ol>
	<li>Check the validity of each separation, by checking that both the current during the separation and the migration time of the electroosmotic mobility marker (DMSO in this case) are similar to the ones observed for the oligoacrylate standard separation. 
	NOTE: Up to 10-15% variation is acceptable from the expected current value of about 50 &#181;A and migration time value of 1.3 min (electrophoretic mobility values should be used instead of migration times if a higher repeatability is required).</li>
	<li>For each successful separation, export the raw data from the capillary electrophoresis software by selecting a specific data set, right clicking on export and selecting an appropriate signal.</li>
	<li>Convert the raw data recorded by the CE (presented as UV absorbance as a function of migration time). Convert the X-axis (migration time tm) into an electrophoretic mobility &#181; following Equation 1: 
	<img alt="Equation 1" src="/files/ftp_upload/54549/54549eq1.jpg" />&#160;&#160;&#160;&#160; (1) 
	where Ld is the length to the detector, Lt is the total length of the capillary, V is the voltage, and teo is the migration time of a neutral specie (the DMSO internal standard in this case)27. 
	Convert the Y-axis of the raw data (absorbance in a.u.) to a distribution of electrophoretic mobilities W(&#181;) following Equation 2:28 
	<img alt="Equation 2" src="/files/ftp_upload/54549/54549eq2.jpg" />&#160;&#160;&#160;&#160; (2)</li>
</ol>
9. Additional Characterization of Peptide-grafted Films18
<ol>
	<li>Insert peptide-grafted chitosan films, rolled around themselves, in a 4 mm solid-state NMR rotor. Fill the rotor with phosphate-buffered saline to swell the films, and close the rotor. Wait for a few hours.</li>
	<li>Analyze the film with 13C NMR spectroscopy18.</li>
</ol>

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Chem. 70, 173-181 (1998).</li><li>Harvey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern Analytical Chemistry. , (2000).</li><li>Oliver, J. D., Gaborieau, M., Hilder, E. F., Castignolles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+robust+determination+of+monosaccharides+in+plant+fibers+in+complex+mixtures+by+capillary+electrophoresis+and+high+performance+liquid+chromatography.">Simple and robust determination of monosaccharides in plant fibers in complex mixtures by capillary electrophoresis and high performance liquid chromatography.</a> J. Chromatogr. A. 1291, 179-186 (2013).</li><li>Oliver, J. D., Sutton, A. T., Karu, N., Phillips, M., Markham, J., Peiris, P., Hilder, E. 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Chem. 407, 2543-2555 (2015).</li><li>Rinaudo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+and+chitosan:+Properties+and+applications.">Chitin and chitosan: Properties and applications.</a> Prog. Polym. 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Rapid Commun. 27, 42-46 (2006).</li><li>Chamieh, J., Martin, M., Cottet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+in+Capillary+Electrophoresis:+Transformation+of+Raw+Electropherograms+into+Continuous+Distributions.">Quantitative Analysis in Capillary Electrophoresis: Transformation of Raw Electropherograms into Continuous Distributions.</a> Anal. Chem. 87, 1050-1057 (2015).</li><li>Maniego, A. R., Ang, D., Guillaneuf, Y., Lefay, C., Gigmes, D., Aldrich-Wright, J. 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The authors declare that they have no competing financial interests.

The simplicity of the protocol described here makes it ideally suited to widespread application. However, particular attention needs to be paid to of the following key steps.
Proper CE instrument preparation
It is important to separate a known standard immediately prior to the separation of unknown samples (as well as at the end of a series of separations) to check the validity of the capillary and instrument on the day. This standard can be an olig...

Free solution capillary electrophoresis (CE) is a technique that separates compounds in solutions based on their charge-to-friction ratio1,2. Charge-to-size ratio is often mentioned in the literature, but this simplification does not apply to polyelectrolytes, including polypeptides in this work, and was also shown not to be appropriate for small organic molecules3. CE differs from other separation techniques in that it does not have a stationary phase, only a background electrolyte (usually a buffer). This allows the technique to be robust in its ability to analyze a large range of samples with complex matrices4 such as plant fibers5, fermentation brews6 grafting onto synthetic polymers7, food samples8, and hardly soluble peptides9 without tedious sample preparation and purification. This is especially significant for complex polyelectrolytes which have dissolution issues (such as chitosan10 and gellan gum11) and therefore exist as aggregated or precipitated in solution and have been successfully analyzed without sample filtration. Further, the analysis of sugars in breakfast cereals involved injecting samples with particles of breakfast cereal samples precipitated in water8. This also extends to the analysis of branched polyelectrolytes or copolymers12,13. Extensive work has also been completed in the development of CE techniques specifically for the analysis of proteins for proteomics14, chiral separation of natural or synthetic peptides15 and microchip separations of proteins and peptides16. Since the separation and analysis take place in a capillary, only small volumes of sample and solvents are used which enables CE to have a lower running cost than other separation techniques including chromatography5,6,17. Since the separation by CE is fast, it allows the monitoring of reaction kinetics. This was demonstrated in the case of the grafting of peptides onto chitosan films for improved cell adhesion18.
Chitosan is a polysaccharide derived from the N-deacetylation of chitin. Chitosan films can be used for various biomedical applications such as bioadhesives19 and cell growth substrates18,20, due to chitosan&#39;s biocompatibility21. Cell attachment to specific extracellular matrix proteins, such as fibronectin, collagens and laminin, is directly linked to the survival of the cells22. Notably, different cell types often require attachment to different extracellular matrix proteins for survival and proper function. Cell attachment to chitosan films was shown to be enhanced through the grafting of fibronectin23; however, preparation, purification and grafting of such large proteins is not economically viable. Alternately a range of small peptides have been shown to be able to mimic the properties of large extracellular matrix proteins. For example, peptides such as the fibronectin mimetics RGD (arginine - glycine - aspartic acid) and RGDS (arginine - glycine - aspartic acid - serine) have been used to facilitate and increase cell attachment24. Covalent grafting of RGDS onto chitosan films resulted in improved cell attachment for cells known to attach to fibronectin in vivo18. Substituting larger proteins likes fibronectin with smaller peptides that have the same functionality provides a significant cost reduction.
Here, peptide grafting to chitosan was performed as previously published18. As previously demonstrated, this approach provides simple and efficient grafting by using the coupling agents EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to functionalize the carboxylic acid of the RGDS to be grafted onto the chitosan film. Two advantages of this grafting method are that it does not require any modification of the chitosan or of the peptide, and it is undertaken in aqueous medium to maximize compatibility with future cell culture applications18,20. As the coupling agents and the peptide can be charged, CE is a suitable method for the analysis of the reaction kinetics. Importantly, analysis of the reaction kinetics via CE enables real-time monitoring of the grafting reaction, and thus enables both optimizing and quantifying the degree of grafting.
While it is not routinely necessary, the results of the CE analysis can be validated off-line by a direct measurement of the peptide grafting onto the chitosan films using solid-state NMR (nuclear magnetic resonance) spectroscopy25,26 to demonstrate the covalent grafting of the peptide onto the film18. However, compared with solid-state NMR spectroscopy, the real-time analysis provided by CE enables the quantification of the peptide consumption in real time and thus the ability to assess the kinetics of the reaction.
The above mentioned method is simple and allows the real-time analysis of peptide grafting onto chitosan films with indirect quantification of the extent of the grafting. The demonstrated method can be extended to the real time quantitative assessment of different chemical reactions as long as the reactants or the products to be analyzed can be charged.

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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Water</td>
			<td style="width:185px;">Millipore</td>
			<td style="width:119px;"></td>
			<td style="width:479px;">All water used in the experiment has to be of Milli-Q quality</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chitosan powder (medium molecular weight)</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td>448877</td>
			<td style="width:479px;">lot MKBH1108V was used. Significant batch-to-batch variations occur with natural products such as polysaccharides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid - Unilab</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td style="width:119px;">2-2.5L GL</td>
			<td>laboratory reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>laboratory reagent, slightly hazardous to skin, hazardous if ingested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465&#160;</td>
			<td>laboratory reagent, corrosive&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RGDS&#160;</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:119px;">H&#8208;1155</td>
			<td>peptide, bought from Auspep Pty Ltd</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-ethyl-3-(3-dimethylaminopropyl)carbodiimide&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">D80002</td>
			<td>Irritant to skin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-hydroxysuccinimide&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">130672</td>
			<td>Irritant to skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride&#160;</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td>466-500G</td>
			<td>laboratory reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride - Univar</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td>384-500G</td>
			<td>analytical reagent, slight skin irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium hydrogen phosphate - Unilab</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td>1234-500G</td>
			<td>laboratory reagent, slight skin irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium dihydrogen phosphate - Univar</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td>4745-500G</td>
			<td>analytical reagent, slight skin irritant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Oligoacrylate standard</td>
			<td style="width:185px;">custom made</td>
			<td style="width:119px;"></td>
			<td style="width:479px;">See reference for synthetic protocol: Castignolles, P.; Gaborieau, M.; Hilder, E. F.; Sprong, E.; Ferguson, C. J.; Gilbert, R. G. Macromol. Rapid Commun. 2006, 27, 42-46</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric acid&#160;</td>
			<td style="width:185px;">BDH AnalR, Merck Pty Ltd</td>
			<td style="width:119px;">10058</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid - Unilab</td>
			<td style="width:185px;">Ajax Finechem</td>
			<td style="width:119px;">A1367-2.5L</td>
			<td>laboratory reagent, corrosivie</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fused silica tubing</td>
			<td style="width:185px;">Polymicro (Molex)</td>
			<td style="width:119px;">TSP050375</td>
			<td style="width:479px;">Flexible fused silica capillary tubing with standard polyimide coating, 50 &#181;m internal diameter, 363 &#181;m outer diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent 7100 CE</td>
			<td style="width:185px;">Agilent Technologies</td>
			<td style="width:119px;">G7100CE</td>
			<td>Capillary electrophoresis instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital shaker&#160;</td>
			<td style="width:185px;">IKA</td>
			<td style="width:119px;">KS260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Electronic balance</td>
			<td style="width:185px;">Mettler Toledo</td>
			<td style="width:119px;">MS204S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Milli-Q Synthesis&#160;</td>
			<td style="width:185px;">Millipore</td>
			<td style="width:119px;">ZMQS5VF01</td>
			<td>Ultrapure water filtration system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Parafilm&#160;</td>
			<td style="width:185px;">Labtek</td>
			<td style="width:119px;">PM966</td>
			<td>Parrafin wax</td>
		</tr>
	</tbody>
</table>

capillary electrophoresis to monitor peptide grafting onto chitosan films in real time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

CE is well suited to monitoring the grafting of peptides (e.g., RGDS) onto chitosan films. Suitable coupling agents include EDC-HCl and NHS which activate the peptide to be grafted onto the chitosan (Figure 1). CE is able to separate the different molecules of interest from the reaction medium. To assign the peaks on the electropherogram, pure RGDS, EDC-HCl and NHS were dissolved, injected and separated separately. After the peak assignment, the reaction medium w...

Watch this Scientific Journal Video about Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time at JoVE.com

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

capillary-electrophoresis-to-monitor-peptide-grafting-onto-chitosan

Research

JoVE Journal

Chemistry

9.0K Views.  Western Sydney University. Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Video: Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements are simple high-throughput characterization experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glassy states for a variety of glassy materials. This technique allows for these parameters to be measured in a single experiment, while other methods must combine a variety of different techniques to investigate all of these properties. Measurements of dynamics close to Tg are particularly challenging. The advantage of cooling rate dependent Tg measurements over other methods which directly probe bulk and surface relaxation dynamics is that they are relatively quick and simple experiments, which do not utilize fluorophores or other complicated experimental techniques. Furthermore, this technique probes the average dynamics of technologically relevant thin films in temperature and relaxation time (&#964;&#945;) regimes relevant to the glass transition (&#964;&#945; &#62; 100 sec). The limitation to using ellipsometry for cooling rate dependent Tg experiments is that it cannot probe relaxation times relevant to measurements of viscosity (&#964;&#945; &#60;&#60; 1 sec). Other cooling rate dependent Tg measurement techniques, however, can extend the CR-Tg method to faster relaxation times. Furthermore, this technique can be used for any glassy system so long as the integrity of the film remains throughout the experiment.

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

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