Proof of concept
LuciPEP aims to detect microplastics in aquatic samples. In order to achieve this, we designed a fusion protein that can bind to polypropylene; NanoLuc luciferase (NLuc) is fused to an anchor peptide binding the plastic of interest. The detection of microplastics depends on the successful binding of the fusion protein with microplastics following multiple washes. The binding is confirmed based on the luminescence intensity detected after adding the substrate luciferin. To facilitate the proof of concept and act as a control, a second fusion protein was designed with eGFP (enhanced green fluorescent protein) instead of NLuc.
Protein production Binding affinity Binding specificity References

Protein production

We were able to successfully clone, express and purify the fusion proteins with eGFP and NanoLuc. We were able to visualise the purified proteins with the help of a plate reader/spectrophotometer by adding the substrate (furimazine) to our NanoLuc fusion protein and adjusting the excitation and emission frequencies for our eGFP fusion protein respectively. Both fusion proteins emitted detectable signals, confirming their accurate folding and supporting our initial construct designs. This allowed us to go forward with the development of our polypropylene binding assay.

Binding affinity

Based on the previous use of the LCI anchor peptide, 1 g of the anchor peptide is enough to bind 654 m2 of polypropylene surface (Rübsam et al., 2017), we decided on a set of different concentrations of our NLuc fusion protein and tested the binding on polypropylene 96 well plates. Post washing, we were able to observe the differences in luminescence intensity. The washing step removes all unbound proteins. Hence, a detectable signal confirms the binding affinity of our fusion protein with the plastic surface.

The luminescence intensity was measured at different concentrations (µg/µl) pre-wash (A) and post-wash (B). Pre-wash luminescence values are higher than that of the post wash values. However, post-wash luminescent signal increases following a threshold protein concentration of 1µg/µl. This indicates the successful binding of our fusion protein.

Binding specificity

An initial test was done to ascertain the binding of the protein construct to polystyrene. No luminescent signal was detected post-wash, supporting the specificity of our construct. However, due to time constraints, a full binding analysis with the appropriate controls, comparable to the one done with polypropolene plates, was not carried out. These results would enable us confirm our binding specificity.

References

Bauten, W., Nöth, M., Kurkina, T., Contreras, F., Ji, Y., Desmet, C., ... & Schwaneberg, U. (2023). Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput. Science of The Total Environment, 860, 160450.