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

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.