Name: capillary electrophoresis to monitor peptide grafting onto chitosan films in real time
Uploaded: 2016-10-26T13:00:00
Description: Watch this Scientific Journal Video about Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time at JoVE.com

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 ...

<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. 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+monitoring+of+ethanol+fermentations+by+capillary+electrophoresis.">Simple and robust monitoring of ethanol fermentations by capillary electrophoresis.</a> Biotechnology and Applied Biochemistry. 62, 329-342 (2015).</li><li>Thevarajah, J. J., Sutton, A. T., Maniego, A. R., Whitty, E. G., Harrisson, S., Cottet, H., Castignolles, P., Gaborieau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+Heterogeneity+of+Chemical+Structures+in+Complex+Charged+Polymers+through+the+Dispersity+of+Their+Distributions+of+Electrophoretic+Mobilities+or+of+Compositions.">Quantifying the Heterogeneity of Chemical Structures in Complex Charged Polymers through the Dispersity of Their Distributions of Electrophoretic Mobilities or of Compositions.</a> Anal. Chem. 88, 1674-1681 (2016).</li><li>Toutounji, M. R., Van Leeuwen, M. P., Oliver, J. D., Shrestha, A. K., Castignolles, P., Gaborieau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+sugars+in+breakfast+cereals+using+capillary+electrophoresis.">Quantification of sugars in breakfast cereals using capillary electrophoresis.</a> Carbohydr. Res. 408, 134-141 (2015).</li><li>Miramon, H., Cavelier, F., Martinez, J., 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=Highly+Resolutive+Separations+of+Hardly+Soluble+Synthetic+Polypeptides+by+Capillary+Electrophoresis.">Highly Resolutive Separations of Hardly Soluble Synthetic Polypeptides by Capillary Electrophoresis.</a> Anal. Chem. 82, 394-399 (2010).</li><li>Mnatsakanyan, M., Thevarajah, J. J., Roi, R. S., Lauto, A., Gaborieau, M., 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=Separation+of+chitosan+by+degree+of+acetylation+using+simple+free+solution+capillary+electrophoresis.">Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis.</a> Anal. Bioanal. Chem. 405, 6873-6877 (2013).</li><li>Taylor, D. L., Ferris, C. J., Maniego, A. R., Castignolles, P., in het Panhuis, M., Gaborieau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Gellan+Gum+by+Capillary+Electrophoresis.">Characterization of Gellan Gum by Capillary Electrophoresis.</a> Australian Journal of Chemistry. 65, 1156-1164 (2012).</li><li>Thevarajah, J. J., Gaborieau, M., 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=Separation+and+characterization+of+synthetic+polyelectrolytes+and+polysaccharides+with+capillary+electrophoresis.">Separation and characterization of synthetic polyelectrolytes and polysaccharides with capillary electrophoresis.</a> Adv. Chem. 2014, 798503 (2014).</li><li>Sutton, A. T., Read, E., Maniego, A. R., Thevarajah, J., Marty, J. -. D., Destarac, M., Gaborieau, M., 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=Purity+of+double+hydrophilic+block+copolymers+revealed+by+capillary+electrophoresis+in+the+critical+conditions.">Purity of double hydrophilic block copolymers revealed by capillary electrophoresis in the critical conditions.</a> J. Chromatogr. A. 1372, 187-195 (2014).</li><li>Righetti, P. G., Sebastiano, R., Citterio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+and+isoelectric+focusing+in+peptide+and+protein+analysis.">Capillary electrophoresis and isoelectric focusing in peptide and protein analysis.</a> Proteomics. 13, 325-340 (2013).</li><li>Ali, I., Al-Othman, Z. A., Al-Warthan, A., Asnin, L., Chudinov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+chiral+separations+of+small+peptides+by+capillary+electrophoresis+and+chromatography.">Advances in chiral separations of small peptides by capillary electrophoresis and chromatography.</a> J. Sep. Sci. 37, 2447-2466 (2014).</li><li>Kasicka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+capillary+and+microchip+electroseparations+of+peptides+(2011-2013).">Recent developments in capillary and microchip electroseparations of peptides (2011-2013).</a> Electrophoresis. 35, 69-95 (2014).</li><li>Taylor, D. L., Thevarajah, J. J., Narayan, D. K., Murphy, P., Mangala, M. M., Lim, S., Wuhrer, R., Lefay, C., O'Connor, M. D., Gaborieau, M., 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=Real-time+monitoring+of+peptide+grafting+onto+chitosan+films+using+capillary+electrophoresis.">Real-time monitoring of peptide grafting onto chitosan films using capillary electrophoresis.</a> Anal. Bioanal. 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. Sci. 31, 603-632 (2006).</li><li>Li, Z., Leung, M., Hopper, R., Ellenbogen, R., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+self-renewal+of+human+embryonic+stem+cells+in+3D+porous+natural+polymer+scaffolds.">Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds.</a> Biomaterials. 31, 404-412 (2010).</li><li>Domard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+30+years+research+on+chitin+and+chitosan.">A perspective on 30 years research on chitin and chitosan.</a> Carbohydr. Polym. 84, 696-703 (2011).</li><li>Shekaran, A., Garcia, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+engineering+of+extracellular+matrix-mimetic+bioadhesive+surfaces+and+implants+for+tissue+engineering.">Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering.</a> Biochim. Biophys. Acta Gen. Subj. 1810, 350-360 (2011).</li><li>Custodio, C. A., Alves, C. M., Reis, R. L., Mano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+fibronectin+in+chitosan+substrates+improves+cell+adhesion+and+proliferation.">Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation.</a> J. Tissue Eng. Regen. Med. 4, 316-323 (2010).</li><li>Boateng, S. Y., Lateef, S. S., Mosley, W., Hartman, T. J., Hanley, L., Russell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+and+YIGSR+synthetic+peptides+facilitate+cellular+adhesion+identical+to+that+of+laminin+and+fibronectin+but+alter+the+physiology+of+neonatal+cardiac+myocytes.">RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes.</a> Am. J. Physiol. Cell Physiol. 288, C30-C38 (2005).</li><li>Lefay, C., Guillaneuf, Y., Moreira, G., Thevarajah, J. J., Castignolles, P., Ziarelli, F., Bloch, E., Major, M., Charles, L., Gaborieau, M., Bertin, D., Gigmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+modification+of+chitosan+via+nitroxide-mediated+polymerization.">Heterogeneous modification of chitosan via nitroxide-mediated polymerization.</a> Polym. Chem. 4, 322-328 (2013).</li><li>Gartner, C., Lopez, B. L., Sierra, L., Graf, R., Spiess, H. W., Gaborieau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Structure+and+Dynamics+in+Chitosan+Films+Investigated+with+Solid-State+NMR,+Dynamic+Mechanical+Analysis,+and+X-ray+Diffraction.">Interplay between Structure and Dynamics in Chitosan Films Investigated with Solid-State NMR, Dynamic Mechanical Analysis, and X-ray Diffraction.</a> Biomacromolecules. 12, 1380-1386 (2011).</li><li>Castignolles, P., Gaborieau, M., Hilder, E. F., Sprong, E., Ferguson, C. J., Gilbert, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+separation+of+oligo(acrylic+acid)+by+capillary+zone+electrophoresis.">High resolution separation of oligo(acrylic acid) by capillary zone electrophoresis.</a> Macromol. 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 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.

<table border="0" cellpadding="0" cellspacing="0" width="1124">
	<tbody>
		<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>
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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. ...

The overall goal of this capillary electrophoresis method is to monitor the grafting reaction of peptides onto chitosan films in real time. This method can help answer key questions in the analytical chemistry and polymer chemistry fields, such as how the reaction is proceeding, and how efficient the grafting process is. The main advantage of this technique is that there is no sample preparation needed, and the reaction mixture can be analyzed in real time.Even though this method can provide insight into reaction processes, it can also be applied towards a system, such as the binding of an anti-cancer drug to a drug carrier, or the digestion of rice or breakfast cereals. Weigh out two grams of glacial acetic acid and complete to 100 milliliters with ultra pure water. Add 100 milliliters of the 2%mass percent acetic acid aqueous solution to 1.7 grams of chitosan powder.Stir for five days with a stirring bar and a magnetic stirring plate at room temperature, either covered with aluminum foil or in the dark. Centrifuge the chitosan dispersion at 1, 076 times g at 23 degrees Celsius for one hour. Following centrifugation, collect the supernatant using a syringe and discard the precipitate.For each film, aliquot 10 milliliters of the chitosan suspension into a nine centimeter plastic Petri dish at room temperature. Leave the films covered to dry for at least seven days. Using scissors, cut the dry films into one by one centimeter squares.The experiment can be paused at this stage. Rinse 10 square chitosan films in five milliliters of PBS for two hours in a Petri dish at room temperature. During this time, prepare and validate the capillary electrophoresis instrument.Prepare a 43.5 centimeter bare fused silica capillary with an internal diameter of 50 micrometers by weakening the polymer outer coating of the capillary at the set length with a blunt utensil, and then snapping the capillary. Create a window for the capillary by using a lighter to burn the polymer coating at 8.5 centimeters from the inlet. After the capillary cools, wipe it clean with ethanol.Then, burn the coating of the capillary at each end for a few millimeters with a lighter. After cooling, wipe it clean with ethanol for a second time. Place the capillary inside the 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. Set the parameters of the method for each separation. In the software menu, select Method, and then Edit the Entire Method.Select the CE tab to edit the CE conditions. Set the temperature, time, voltage, and vials used for the separation. In the Preconditioning section, set the consecutive flushes.Use 10 minutes with one molar sodium hydroxide, five minutes with 0.1 molar sodium hydroxide, five minutes with ultra pure water, and five minutes with 75 millimolar sodium borate buffer at pH 9.2 for the first method in the sequence. Check the others methods in the sequence following the same procedure. For the subsequent methods, set the consecutive flushes in the Preconditioning section as one minute with one molar sodium hydroxide, and five minutes with 75 millimolar sodium borate buffer at pH 9.2.In the Injection section, set parameters for a hydrodynamic injection with 30 millibar pressure for 10 seconds for all methods. In the Separation section, set the separation conditions to 30 kilovolts, at 25 degrees Celsius for nine minutes for all methods. Inject and separate a neutral internal standard.For the neutral internal standard, use 10 microliters of 10%volume to volume DMSO in water diluted into 450 microliters of 75 millimolar sodium borate buffer. Before starting the sequence, ensure the sequence parameters are set to the correct folder and file number. Then press Sequence to start the experiment.The separation of the standards allows validation of the instrument before an experiment is run. This is essential for a successful experiment. Then inject and separate an oligoacrylate standard in the same manner to check the validity of the capillary.Pause the sequence here until the grafting reaction is ready to start. To perform grafting of RGDS onto chitosan film, first weigh out the peptide and the coupling agents. Two hours after the start of the chitosan film soaking in PBS, dissolve the peptide and the coupling agents in five milliliters of PBS.Take a 50 microliter aliquot of this solution and add two microliters of 10%volume to volume DMSO in water as an internal neutral standard. Analyze the aliquot with capillary electrophoresis using the same analysis conditions as before. Next, remove the PBS used to rinse the chitosan films from the Petri dish.Add the five milliliter solution of peptide and coupling agents to the Petri dish containing the chitosan films. Cover the Petri dish with paraffin film and place it on an orbital shaker at room temperature. Take 50 microliter aliquots of reaction medium at set times.The aliquots taken from the reaction medium need to be analyzed immediately. The appropriate steps must be taken in quick succession to allow real time analysis. Add two microliters of 10%volume to volume DMSO in water as an internal neutral standard to each aliquot.Analyze aliquots with capillary electrophoresis as soon as they are taken. Upon completion of the separations, rinse the capillary with ultra pure water for 10 minutes. After four hours of shaking and aliquot removal, remove the Petri dish from the shaker.After removing the reaction medium from the Petri dish, add five milliliters of PBS to rinse the chitosan films. Remove the PBS from the Petri dish, rinse the chitosan films with ultra pure water, and allow them to dry overnight. Store the films at minus 20 degrees Celsius in a plastic Petri dish.Representative results of the first aliquot of the chemical reaction show a separation of the different chemical reactants. The RGDS peptide peak at this time is the starting point of the reaction, and can be used to quantify grafting efficiency. As the grafting reaction is continuously monitored, the area of the RGDS peak continues to decrease until it remains constant, which signifies the end of the experiment.A second peak on the right of the EDC HCl peak appears as is expected when the reaction proceeds. An integration of the RGDS peak over time allows the peptide consumption to be calculated. Swollen state NMR spectroscopy of chitosan films in PBS allows the detection of the grafting of RGDS, chitosan film, and chitosan film grafted with the peptide RGDS, or swollen in PBS.The asterisk represent the signals assigned to RGDS. Once mastered, this technique can be completed in six hours, if it is performed properly. While attempting this procedure, it's important to remember to prepare all solutions ahead of time.Following this procedure, other methods like solid state animal spectroscopy can be performed in order to answer additional questions, like the direct validation of the graphing experiments. After watching this video, you should have a good understanding of how to use free solution capillary electrophoresis in the monitoring of chemical reactions.

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