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+ | <h3>Methods</h3> | ||
+ | <h1>Material:</h1> | ||
+ | <p> | ||
+ | Chitosan (high molecular weight, 310-375 kDa, >75% deacetylated), succinic anhydrite, ala-ala-phe-7-amido-4-methylcoumarin, | ||
+ | 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-Hydroxy-2,5-pyrrolidindion were purchased from Sigma-Aldrich. | ||
+ | Acetic acid and Acetone were given from AK Fessner (TU Darmstadt). <br> | ||
+ | We used 250 mL-Dreihalskolbenrührapparatur with a dropping funnel, a spin coater and laboratory shakers.<br> | ||
+ | Laboratory and Lab equipment were provided by AK Fessner and AK Kolmar (TU Darmstadt) and the centrifuge was provided by AK Hausch. Spin-coating was performed in the Laboratory of Prof. Koeppl (TU Darmstadt). | ||
+ | </p> | ||
+ | <h1>Preparation of N-Succinic Chitosan (NSC)</h1> | ||
+ | <p>Aqueous Acetic acid solution was put in the Dreihalskolbenrührapparatur. Chitosan was added in small doses through the flask neck under heavy mechanical stirring. | ||
+ | Therefore a lab stirrer was needed, not just a stir bar, because the solution becomes very viscous. | ||
+ | The solution was stirred until becoming a homogenous semi-fluid. | ||
+ | Now 20 mL of a 1% succinic anhydrite solution in acetone were prepared and added drop-wise to the reaction flask under heavy mechanical stirring for 30 minutes. | ||
+ | The reaction mixture was left overnight at room temperature. The following day, it was centrifuged to separate the precipitate from the left, non reacted Chitosan.</p> | ||
+ | <p>Spin coating is a suitable technique to prepare thin layers out of viscose fluids. In principle, a spin coater is a device where a surface you want to coat is | ||
+ | clamped onto a centrifuge. Through fast rotation of the surface, a fluid is distributed evenly. | ||
+ | We used a microscope slide as surface and coated it with our | ||
+ | Chitosan/N-Succinic Chitosan mixture from the previous step. To determine the best fluid properties we performed four test with different amounts of chitosan | ||
+ | and acetic acid concentrations. Every coating step with the spin-coater was performed at 400 rpm for 20 seconds and then 2000 rpm for 60 seconds.</p> | ||
+ | <h1>Succinyl saturation of Chitin</h1> | ||
+ | <p>To ensure a sufficient amount of N-Succinic Chitosan in our product we performed a second succinylation of the thin-layer. For this step, we tested two different procedures.<br> | ||
+ | <b>1. Succinic anhydride in Acetone</b><br> | ||
+ | We dissolved succinic anhydride in acetone and shook it at 37°C for x hours.<br> | ||
+ | <b>2. Succinic anhydride in DMSO</b><br> | ||
+ | We dissolved succinic anhydride in DMSO and shook it at 60°C for x hours. | ||
+ | </p> | ||
+ | <h1>Grafting of Ala-Ala-Phe-7-Amido-4-methylcoumarin (AAP-AMC) to N-Succinic Chitosan</h1> | ||
+ | <p>The linkage of the AAP-AMC was performed in two steps. First 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was dissolved in methanol and | ||
+ | 1-Hydroxy-2,5-pyrrolidindion (NHS) was added. A separate solution of AAP-AMP in methanol was prepared. The immobilized NSC film was immersed in the EDC/NHS solution | ||
+ | for 60 minutes while shaking. The film was rinsed with methanol. Afterwards, the NSC film was immersed in the AAP-AMP solution for 60 minutes. | ||
+ | The film was rinsed | ||
+ | again with methanol and immersed in methanol for 60 minutes, the methanol was replaced every 15 minutes. | ||
+ | The film was rinsed for the last time with methanol an dried.</p> | ||
+ | </div> | ||
+ | </section> | ||
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Revision as of 18:23, 13 October 2017