The last few years have shown promising developments in single-molecule protein sequencing using a biological nanopore. Using only one membrane pore in a salt buffer, an applied potential creates a measurable ion current through the pore’s constriction. If a small molecule passes through the pore, this disruption is visible as a characteristic current pattern based on the molecular structure of the molecule. Recent developments by Henry in 2021 show that single amino acid substitution in short proteins can be detected using this method. Unfortunately, due to the complexity of the reading, de novo sequencing does not yet apply to these single molecule reads. Nevertheless, detecting different variants of a protein is possible. In current work this method is being developed to detect phosphorylation sites on a peptide of biological significance. For the latter, an immunopeptide called IRS2 is chosen to be once and twice phosphorylated. This phosphorylation causes the peptide to be a cancer biomarker. If this low-cost method of post-translational modification detection is successful, it would be an improvement compared to the standard mass spectrometry sequencing approach. In this BEP research, problems faced before and after sequencing the IRS2 peptide are approached from two different perspectives. The first aim is to adapt the data acquisition software to automate the workflow. The second aim is to adapt and model the Freely Jointed Chain (FJC) Model to a heterogeneously charged peptide. Both aims are defined to be applicable to other peptides as well. For the first aim, a LabVIEW plugin was developed using insights from data processing in MATLAB. This tool detects the state of the nanopore reading, responds using voltage control in a closed feedback loop and frequently allows for calibration checks. All features were tested with training sequences from real data and show promising results. However, more testing in the lab is required to determine its accuracy compared to a human operator. For the second aim, the FJC model was analytically adapted to the IRS2 peptide inside the nanopore’s electric field. The energetically most favourable configuration was then sought with a Metropolis Algorithm. As a result, the electrostatic potential was calculated and implemented into the Metropolis Algorithm. Despite the simplification of this method, it is still expandable in a modular way to incorporate additional potential such as charge-charge interactions or springs to simulate backbone flexibility. Finally, improving this model further would eventually lead to the relative positions of all chain elements inside the pore to develop an understanding of the (phosphorylated) IRS2 readings. ... Read more

Following the perspective of the European Horizon 2020 project ‘NUCLEUS’, Responsible Research and Innovation (RRI) should be brought to life in universities and scientific institutions. This study acts as one of the mobile nuclei of the NUCLEUS project, with the goal to operationalize RRI instead of building theories. The aim of this research is to stimulate reflexivity, one of the dimensions of RRI, amongst scientists in fundamental research within the TU Delft, using a simple tool or method. This is done using a self-developed co-design approach, based on design-based research. This research is the first attempt to create a tool together with the end-users, the fundamental scientists, to stimulate RRI.

In this study, the mirror tool is designed and tested. On micro-ethics level (first order) reflexivity was stimulated using the tool. Especially first year’s students mentioned that the mirror form would help guiding them when reflecting on large projects or when being stuck. However, macro-ethics (second order reflexivity) was not considered using the tool, and thus the socio-ethical and socio-economic aspects were not taken into account yet. Therefore, changes to the design are proposed assuring also macro-ethics will be considered.

Using the tool, not only reflexivity is stimulated, but also responsiveness is encouraged, due to the last question ‘So what?’. In this way, not only learning and thinking will change, but also adjustments can be made to the current way of working.

Implementation of the tool at the BN department of the TU Delft could be done via two ways: either via the graduate school or via the principle investigators. Furthermore, quantitative tests should be performed to statistically prove the effect of the tool, increasing its reliability. Besides, the tool could be more functional when digitalizing the mirror form, such that users are not limited by the amount of writing space and such that supplementary elements could be implemented.

Apart from using the mirror tool in their universities, the NUCLEUS consortium can additionally benefit from the new methodology to create a tool for RRI with and for scientists. Using the one page guideline (appendix 17), consortium members can conveniently create a tool for RRI for their specific target group and in their context. ... Read more