The research conducted as a part of my PhD studies at the Institute of Organic Chemistry, Lodz University of Technology (Poland), focuses on developing a novel bio- detection system indicating disease entity (especially COVID-19) by recognition of characteristic compounds present in physiological fluids e.g. blood, urine or exhaled air with saliva particles. The concept of study is based on the preparation of a biosensor with an immobilized active entity, responsible for determining the SARS-CoV-2 virus particles in the patient’s breath or the air. One option is using a human angiotensin-converting enzyme type 2 (hACE2), described as receptor binding the spike protein subunit of the novel coronavirus with high affinity. Immobilization of the hACE2 on a nanogold-covered sensor through thiol chemistry would allow for observation of the interaction with coronavirus particles and generate a signal delivering qualitative and quantitative information about COVID-19 disease, without the necessity to extract analyte from biological mixture.
Concerning the fact that maintaining a biosensor including the entire protein structure would lead to difficulties resulting from problematic, expensive and time-consuming handling of a protein membrane, a promising alternative involved using smaller fragments of hACE2 due to their smaller size, higher stability and temperatures and pH changes or facilitated sterilization and storage conditions.
Searching peptide structures with potential biological activity that recreate the outer sphere of the protein and are involved in protein-peptide or protein-protein interaction based on the Dot-blot methodology. The initial phase of the research included the automated SPOT synthesis of overlapping decapeptide fragments covering the whole primary structure of the hACE2 in a cellulose matrix using triazine-based coupling reagents. Obtained 399-elemented library was incubated with the HRP-labelled S1 protein subunit of the SARS-CoV-2 to detect structures forming colourful complexes. Based on their colour intensity, 32 peptide fragments were selected with a different length of peptide chain, in which 16 were characterized as products strongly interacting with spike protein.
The collaboration with the interdepartmental research unit of Peptide and Protein Chemistry and Biology “PeptLab” at the University of Florence (Italy) greatly expanded the possibilities for my current study. Projects led by team members concerning the use of peptides in functionalized materials, and the development of SPR-based biosensors detecting biomolecular interaction or peptide inhibitors for COVID-19 were crucial for me during the process of searching for a Host Institution.
The experimental work during the two-month internship included:
• Automated synthesis of 7 peptide fragments of hACE2 protein, characterized by the highest colour intensity in the Dot-blot test, based on the SPPS methodology and Fmoc/t-Bu strategy with an additional induction-heating and real-time UV monitoring. The value added was the application of a more environmentally friendly mixture of ethyl acetate and dimethyl sulfoxide instead of more toxic DMF solvent;
• Purification of obtained products from minor impurities using flash and/or semi-preparative chromatography method;
• Continuous examination and characterization of obtained products using UPLC-MS technique;
• Studying molecular interactions between S protein of the SARS-CoV-2 virus (ligand)
and synthesized hACE2 peptide fragments (analytes) in surface plasmon resonance technique (SPR), which included establishment of suitable conditions for ligand immobilization, covalent immobilization of a spike protein on dextran-modified gold surface of sensor based on EDC/NHS chemistry and first screening of the interaction between selected peptide fragments and S protein.
Training at the University of Florence provided me with an excellent opportunity to gain in-depth knowledge of peptide chemistry. Additionally, I learned a new method for examining peptide-peptide and peptide-protein interactions, which will help me in my ongoing study concerning the SARS-CoV-2 virus. Furthermore, the internship substantially contributed to my academic and personal development by enhancing soft skills (teamwork, communication, adaptability) and language skills.
Moreover, the fellowship has facilitated the significant potential for further international work between the Lodz University of Technology and the University of Florence. Experimental work concerning the use of hACE2 fragments for COVID-19 identification will be continued. The first attempts suggested 2 peptide fragments that can maintain biological activity towards the S protein of the SARS-CoV-2. Further plans for the study also include: examining the interaction between spike protein and other hACE2 peptide fragments to estimate their kinetics and binding affinity, computer simulations exploring possible interactions of selected peptides with spike protein, conformational studies for chosen structures using circular dichroism or biological studies.
