Team:TU Darmstadt/Demonstrate

ChiTUcare

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Proof of Concept

Here we want to give you a short tour about what we achieved in the project. We show our results and give a view on the resulting product, a protease-sensing hydrogel.

Chitin Synthase NodC

We wanted to produce chitin as precursor for our designer Chitosan in E. coli, using the chitin synthase NodC. We were able to express the chitin synthase NodC in E. coli Top10 successfully. The protein was tagged with a His‑tag and purified via an ÄKTA system. To verify the expression and purification a SDS-PAGE was done.
The functionality of the NodC enzyme was verified by performing the UDP-Glo™ Glycosyltransferase Assay. The evaluation of the assay shows that the NodC enzyme converts the UDP-GlcNAc to free UPD and a growing oligo‑GlcNAc‑chain. The free UDP is converted to ATP, which acts as a substrate for a luciferase reaction and creates luminescence. So the assay and the increasing luminescence depending on increasing enzyme concentrations shows that the NodC enzyme can create chitin oligomers.
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Figure 1. Activity assay of NodC. The NodC (40 ng) was titrated in 1X glycosyltransferase reaction buffer in the presence of 100 μM of UDP-N-acetylglcosamine and 10 mM N-acetylglucosamine (GlcNAc) as an acceptor substrate. The reaction was performed as described before and the luminescence was measured after 1 hour of incubation with a Tecan200 Infinite Pro plate reader. Each point is an average of two experiments, and the error bars represent the standard deviations. RLU = relative light units.

Chitin Deacetylase NodB

In order to produce our designer chitosan as a wound healing improving material in our hydrogel, we used the chitin deacetylase NodB. We verified the expression of NodB in E. coli BL21 with a SDS-Page. After tagging the protein with a His-Tag, we performed purification and refolding through an ÄKTA pure system, and verified the success through another SDS-Page.
To test whether NodB works properly, we used the acetic acid assay kit (Acetate Kinase Manual Format, Megazyme, Bray, Ireland). NodB deacetylates chitin to create chitosan. This chemical step releases acetic acid. The amount of acetic acid is indirectly measured via amount of NAD+. Thus, the rate of acetic acid is stoichiometric with the amount of NAD+ in the last reaction step. As the graph shows, this verification was successful and indicates that NodB was refolded properly and is present in its active form.
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Figure 2. Activity Assay of NodB. Detected acetic acid after 76 minutes in 20 %, 15 % and 10 % sample volume generated by NodB in 60 % standard solution. Four samples with 1 mM chitin pentamers and 2.5 µM purified NodB were previously incubated overnight in NH4HCO3 adjusted to pH 9 at 37°C. Afterwards these were heat-inactivated for 20 minutes at 80 °C. The measurement with Tecan200 infinte Pro plate reader ranged over 76 minutes.

Hydrogel

Supposing to build an ideal scaffold for our smart protease-sensing bandage, we manufactured several hydrogels containing non-toxic and cost-effective gelling agents. Our hydrogels could be formed with basic laboratory equipment at any shape and could easily be adjusted to the affected tissues for optimal wound healing.
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Figure 3. We manufactured an agar-chitosan hydrogel. Using basic laboratory equipment as well as cheap and non-toxic reagents, we produced a row of different chitosan containing hydrogels. They can be used as a basis for a medically applicable wound cover.

Chemistry

In the interest of adding the abillity to detect pathogenic bacteria to our hydrogel, we manufactured a protease‑sensing chitosan derivative. Therefore, we immobilized a quenched flourophor via a specific peptide linker. For the verifaction of its functionality it was measured via a fluorometer. It showed a peak at 390 nm before the protease was added. After cleavage with proteases, a shift of this peak to the wavelength of 450 nm was observed.
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**Figure 5. We engineered a protease detecting chitosan derivative.** Left: Left: Fluorometric measurement of the blank, diluted peptide solution (8 mMol), and protease treated peptide solution. Right: Diluted peptide solution (8 mMol), and protease treated peptide solution exposed to UV light (366 nm).

ChiTUcare

To show that our project works, we manufactured a chitosan hydrogel with a fluorophore coupled to it. Upon addition of a protease solution to our ChiTUcare prototype, the hydrogel starts to glow under UV light within 7 minutes (the video speed was increased).
This proves our concept of a protease sensing hydrogel on the basis of chitosan.
As an easy and cheap to manufacture prototype, we provide an eco-friendly and adjustable system to nurture wound-healing with an antibacterial, anti-viral wound dressing, that works as a sensor for bacterial infection by emitting a blue light within minutes upon irradiation of non-hazardous UV light.