Introduction

Badly healing wounds are still a big issue in clinical medicine all over the world. Especially inflamed wounds often exhibit impaired healing properties and are prone to infections of opportunistic pathogenic bacteria. On the one hand, wounds have to be screened for infections extensively, on the other hand, they have to be kept wet and in an oxygen-free atmosphere for optimal healing conditions. Thus there is an obvious contradiction between the best healing conditions and the commonly used infection swab test, where the wound coating has to be removed. Furthermore, current swab tests need a few days to evaluate the presence of pathogenic bacteria, but it is important to get this information as soon as possible and to start the suitable treatment. Ebrahimi and Schönherr developed a quick and non invasive detection method for wound infections without the necessity to remove the wound coating. The principle of the test is the modification of an amino group of chitosan with succinic anhydride to gain a carboxyl group that can be linked to the amino group of our alanyl-alanyl-phenylalanine peptide linker. This peptide linker is fused to our actual detectable unit, the 4-methylcoumarin.

This linker was chosen due to the fact that chymotrysin cleaves peptides N-terminal of aromatic amino acids, so here it would cleave N-terminal of phenylalanin.

In our studies, we present an uncomplicated method to repeat the work of Ebrahimi and Schönherr in a way that works for iGEMers and FabLabers. For that, we combined a few instructions to guarantee a working product without the need of expensive instrumental analysis.

Methods

Material:

Chitosan (high molecular weight, 310-375 kDa, >75 % deacetylated), succinic anhydride, 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).
We used 250 mL-three-necked flask with a dropping funnel, a spin coater and laboratory shakers.
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).

Preparation of N-Succinic Chitosan (NSC)

Preperation of saturated N-Succinic Chitosan-Hydrogel

To increase the saturation of the succinic-chitosan binding we proceeded with the step where succinic anhydride in acetone was added twice.

Preparation of thin-layer Chitosan/N-Succinic Chitosan

Spincoating is a suitable technique to prepare thin layers out of viscous fluids. In general, a spin coater device works as following: Firstly, a surface is clamped onto a centrifuge. Through fast rotation of the surface, a fluid is then distributed evenly to create the coating. 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 tests 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.

Succinyl Saturation of the Chitosan-Film

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.
1. Succinic anhydride in Acetone
2.5 g succinic anhydride were solved in 100 mL aceton in a glas petri dish. Our microscope slide with the thin-layer chitosan/N-succinic chitosan was placed inside. The petri dish then was sealed with parafilm and shaken for 72 hours at 37 °C. The evaporated aceton was replaced every 12 hours
2. Succinic anhydride in DMSO
We put 50 mL DMSO and 2.5 g succinic anhydride in a closable glass and and shook it for 2 hours at 60 °C. After the succinic anhydride was completely dissolved we placed our microscope sildes inside the glass and continued shaking at 60 °C for 24 hours.

Grafting of Ala-Ala-Phe-7-Amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to the N-Succinic Chitosan-Film

The linkage of the Ala-Ala-Phe-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 Ala-Ala-Phe-AMC 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 Ala-Ala-Phe-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.

Grafting of Ala-Ala-Phe-7-Amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to the N-Succinic Chitosan-Hydrogel

Due to the fact that the saturated N-succinic chitosan-hydrogel is almost completely soluble in water it is possible to carry out the coupling in a liquid phase. 100 mL of the hydrogel were solved in the same amount of water. Subsequently EDC (~ 1 mol: 1 mol COOH) and NHS (~ 1 mol: 1 mol COOH) were dissolved in methanol, as well as Ala-Ala-Phe-7AMC in a different solution. The EDC/NHS solution was added to the reaction flask. After 60 minutes, while shaking, the Ala-Ala-Phe-7AMC solution was added. After another hour under shaking, the hydrogel was fast filtered and dried at room temperature.

Spin Coat a thin layer of Ala-Ala-Phe-7-Amido-4-methylcoumarin-N-Succinic-Chitosanhydrogel

The hydrogel could either be dried at room temperature as described in the last entry, but it can also be spincoated on another hydrogel to save material. For this purpose the hydrogels prepared by the hydrogel team were used. The compounds of hydrogel was a 1:1 mixture of Agar and Chitosan in acetic acid.

Verification of the Ala-Ala-Phe-7AMC and the successful coupling to the Film and the Hydrogel

Fluorescence Emission via Fluorometer

To verify that the protease has actually cleaved the 7-AMC from the peptide we checked the fluorescence emission of the solved peptide in methanol before and after the treatment with the protease. The Ala-Ala-Phe-7AMC was solved in methanol and protease was added. The film was dipped in a protease solution and checked after 15 min. The hydrogel was moistened with protease solution and checked after 5 minutes. The 7-AMC has an excitation wavelength of 325 nm, its emission wavelenght lies at 450 nm, therefore in the visible spectrum. The used protease was bovine chymotrypsin.

Fluorescence Emission via naked Eye

For the cleavage we used the bovine chymotrypsin. The dissolved peptide was put in a cuvette and protease was added. The fluorescence became visible with an ordinary UV lamp. The film was dipped in a protease solution and also observed using an ordinary UV lamp. Last but not least we moistened the hydrogel with protease solution and observed it using an ordinary UV lamp again.

Results

The different Gels

To determine the best combination of amounts and concentrations for the preparation of the NCS film and hydrogel we produced five different gels with different characteristics.

The first gel with the concentration of wt 1 % of the acetic acid showed a semi stable consistency and was really sticky on glassy surfaces. Due to this characteristics it was very easy to spincoat a flat even film on a microscope slide with it.

The second gel with the concentration of wt 0.5 % showed a more liquid consistency then the first one but still was stable enough to form a hydrogel. When it came to the spincoating the film, it appeared, that the thin layer was too dry and could not form a hydrogel. Moreover it seemed that the N-succunic chitosan crystalized on the microscope silde, meaning that we could not modify it as a hydrogel at all.

The third gel with the concentration of wt 2 % showed a much more stable and solid consistency then the first and the second gel. Nevertheless, we were able to spincoat it on a microscope slide, although the layer was not as even as the first gel.

The fourth gel with the concentration of 1 % but only 0.7 g of chitosan did not create a gel. It was just a liquid containing chitosan-gel fragments. It was not possible to spincoat a film with this solution.

The fifth gel with the concentration wt 1 %, where the succinic anhydride coupling was done twice, formed a stable film that could be dissolved in approximately double the amount of water. If it is dried out it forms a hydrogel again. When moistened with water, it swells again. This procedure is repeatable. With the water you may be able to control the consistency. If the gel is spin coated the best ratio of water and gel is 1:1. The film is even and very stable. It is even possible to form a thick gel layer, if it is dried at room temperature overnight.

Films №1, №3 and №5 were used for further treatment.

The Fluorescence Emission

Measurement via Fluorimeter

The Ala-Ala-Phe-7AMC/methanol solution showed a peak at 390 nm before the protease was added. After the cleavage with the protease a shift of this peak to the wavelength of 450 nm was observed. All the measurements were repeated with different concentrations of Ala-Ala-Phe-7AMC and protease. It could be observed, that with decreasing concentration of the Ala-Ala-Phe-7AMC the intensity of the fluorescence also decreased. And with decreasing concentration of the protease the effect of the digestion got lower until it was almost undetectable within a time, that is suitable for the fast detection.

Measurement via naked Eye

The Ala-Ala-Phe-7AMC/methanol solution in the cuvettes with and without protease was placed on an ordinary UV lamp table. It came to the result that it is possible to observe fluorescence that is caused by proteases in a concentration, that is common for inflamed wounds (up to 500 µg) [2]. A leading role of the visible feedback at such a low concentration has the concentration of the peptide in the gel. At a concentration of 0.1 µg/ml it is still easy for the naked eye to see the fluorescence at a protease concentration of 500 µg.

Considering the outcome of the measurement of the dissolved peptide the films and hydrogels confirmed the results. The detection is completed in 5 to 10 minutes and it is easy to spot for the naked eye to see the cleavage of the fluorescence dye under an ordinary UV lamp. In addition to that it enough to just coat a thin layer of this hydrogel on a surface or another hydrogel to still have this strong effect.

It is possible to form a cheap, reliable enzyme detection system for proteases out of a chitosan-Hydrogel that is able to detect the proteases in 5 minutes.

Conclusion

purpose of that study was to provide principle technique for the production of an enzyme-sensing hydrogel out of biosynthetical produced chitosan , which is reliable and capable even for low budget laboratories. Especailly the cheap production of the chitosan layers with the peptide that are capable to detect pathogenic bacteria in wounds was a hugh success. We offer the iGEM community our method for free use.
In the first experiments we had some struggle to form a hydrogel that showed the wanted characteristics. That the hydrogel wasn’t dissolvable in water was first a problem, but after we consulted Prof. Schoenherr from the university of Siegen we decided to spin coat the produced hydrogels and modify just the layers of the gels. This worked very fine and the results were very encouraging to keep on going. So we decided to form a full thick hydrogel that should be able to detect proteases. For this puropose we get back to the succinic modification of the whole hydrogel not just the layer. And after some trials we were capable to saturate a hydrogel with the succinic anhydride. This hydrogel was partly solutable in water and so the coupling of the peptide was even easier than the surface modification. After the desiccation the gel showed perfect characterics to be used as easy way to detect wether there are protease in a solution or not. Another advantage is that the whole detection reaction just need 5 Minutes to detectable with the naked eye. If we apply these results to our problem of the infected wounds the produced hydrogel represent a easy cheap and simply usable solution for a fast detection. Compared to the standard methods infection detection the hydrogel is better for the wound, much much faster and usable for everybody.

Acknowledgements:

We would like to thank Prof. Dr. Schönher from the University of Siegen for helping us with the problems in our early project. We also thank Prof. Dr. Kolmar and Prof. Dr. Fessner for the opportunity to use their Laboratories. Additionally, we thank Marie-Luise Reif for supporting us in the chemical Lab. We express our special gratitude to Dr. Avrutina who helped us with many different issues and always had an ear for our problems and missing chemicals and enzymes. In addition to that, we want to thank Prof Koeppl for using his Spin Coater and Francois-Xavier Lehr and Tim Prangemeier for showing us how to use it.

References

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