PCDA Synthesis and Polymerisation

Our detection method is based on PCDA polymer vesicles that are able to change colour when subjected to various kinds of stress, such as chemical, mechanical, heat and radiation.

The first step was to synthesise PCDA polymers from crystalline PCDA monomers. In their polymer form, PCDAs adopt vesicle formations in a water solution. This solution can then be stored at 4 ℃ for extended periods of time.

We tested the PCDA solution for reactivity to heat and UV-radiation. For heat, a few mL of the solution was poured into a glass test tube and subjected to a burner flame. After a few seconds, the solution turned to a bright orange, indicating a structural change. When it comes to UV-radiation, the colour change is a lot more subtle, changing to a deep purple when left overnight under UV-radiation. This is probably due to the UV lamps’ supplied energy being relatively low, especially compared to the burners energy flux into the solution.

Figure 1: Colour changes showing a structural change

Spectrophotometric measurements of the colour change were performed and the absorbance maxima changes from around 640 nm to around 500 nm. Both peaks visible on the spectrum are shifted to lower wavelengths when the PCDA solution is heated.

Figure 2: Spectrometric absorbances

PCDA - Protein A Aptamer Conjugation

The next step was to conjugate our PCDA with the aptamer and then test it using protein A. After incubation of the protein A with the PCDA-aptamer conjugate we did not detect any color change so we tested if the protein A aptamer is actually binding protein A. We examined that in 3 different ways:

• Protein A coated beads
• Chromatography using a protein A coated column
• Binding assay using a fluorescent protein A aptamer

Unfortunately, the first two ways, beads and chromatography, yielded no positive results (images 3,4). However, the binding assay kinda worked.

3 IMAGES HERE

SELEX

Another crucial part of our project was to create the two aptamers, one for the lipoteichoic acid and one for c. acnes, using SELEX. We faced a lot of problems during this process. The main obstacle was the optimization of the PCR. From wrong concentrations of PCR reagents to contaminations and wrong primers we spent a lot of time troubleshooting in order to make the PCR work since it is the most important part of the SELEX process. Everything is written in detail in the engineering and in our troubleshooting guide.

IMAGE HERE

In our last days in the lab we got positive results for our first round of SELEX, which means that, since SELEX is a repetitive process, not only our protocol works but also if we had more time we would probably be able to have successful rounds of SELEX.

Cloning

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References

1. Allen, M. J., & Sheridan, S. C. (2015).

   Mortality risks during extreme temperature events (ETEs) using a distributed lag non-linear model.

   International Journal of Biometeorology 62(1), 57-67.

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   Investigating the impact of influenza on excess mortality in all ages in Italy during recent seasons (2013/14-2016/17 seasons).

   International Journal of Infectious Diseases 88, 127-134.

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3. Ingalls, B. P. (2013).

   Mathematical modeling in systems biology: An introduction.

   MIT Press.

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4. Agriculture: Crop production: Sugarcane. TNAU Agritech Portal.

   (March 15, 2019). Retrieved on June 22, 2020. from https://google.com

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   Agriculture: Crop production: Sugarcane. TNAU Agritech Portal.

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