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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

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

Published: October 26th, 2016

DOI:

10.3791/54549

1Molecular Medicine Research Group, Western Sydney University, 2Australian Centre for Research on Separation Science, Western Sydney University, 3School of Science and Health, Western Sydney University, 4School of Medicine, 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.

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'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.

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 fibers

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1. Preparation of Chitosan Films

  1. Weigh out 2 g of glacial acetic acid, complete to 100 ml with ultrapure water.
  2. 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.
  3. Centrifuge the chitosan dispersion at 1,076 x g at 23 °C for 1 hr. Collect the supernatant with a syringe and discard the precipitate.
  4. For each .......

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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.......

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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.......

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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 Hervé Cottet (Montpellier) for discussions.

....

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Name Company Catalog Number Comments
Water Millipore All water used in the experiment has to be of Milli-Q quality
Chitosan powder (medium molecular weight) Sigma-Aldrich 448877 lot MKBH1108V was used. Significant batch-to-batch variations occur with natural products such as polysaccharides
Acetic acid - Unilab Ajax Finechem 2-2.5L GL laboratory reagent
Dimethylsulfoxide Sigma-Aldrich D4540 laboratory reagent, slightly hazardous to skin, hazardous if ingested
Sodium hydroxide  Sigma-Aldrich 221465  laboratory reagent, corrosive 
RGDS  Bachem H‐1155 peptide, bought from Auspep Pty Ltd
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  Sigma-Aldrich D80002 Irritant to skin 
N-hydroxysuccinimide  Sigma-Aldrich 130672 Irritant to skin
Sodium chloride  Ajax Finechem 466-500G laboratory reagent
Potassium chloride - Univar Ajax Finechem 384-500G analytical reagent, slight skin irritant
Disodium hydrogen phosphate - Unilab Ajax Finechem 1234-500G laboratory reagent, slight skin irritant
Potassium dihydrogen phosphate - Univar Ajax Finechem 4745-500G analytical reagent, slight skin irritant
Oligoacrylate standard custom made 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
Boric acid  BDH AnalR, Merck Pty Ltd 10058 Corrosive
Hydrochloric acid - Unilab Ajax Finechem A1367-2.5L laboratory reagent, corrosivie
Fused silica tubing Polymicro (Molex) TSP050375 Flexible fused silica capillary tubing with standard polyimide coating, 50 µm internal diameter, 363 µm outer diameter 
Agilent 7100 CE Agilent Technologies G7100CE Capillary electrophoresis instrument
Orbital shaker  IKA KS260
Electronic balance Mettler Toledo MS204S
Milli-Q Synthesis  Millipore ZMQS5VF01 Ultrapure water filtration system
Parafilm  Labtek PM966 Parrafin wax

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