Periodic Reporting for period 1 - ENDONANO (Quantitative detection of bacterial endotoxin by novel nanotechnological approaches)
Reporting period: 2019-01-01 to 2020-12-31
All of the fourteen ESRs have been successfully recruited and have been enrolled in Medical Biology PhD Program of Paris-Lodron University Salzburg, one of academic partners. Due to a huge amendment that had to be implemented at the very beginning of the project and restrictions due to Covid-19 pandemic, the ESRs have started their research activities with some delays and difficulties. However, they have collected preliminary results on their individual projects. The research activities mainly addressed: 1. Interaction of endotoxin with biological entities/molecules; 2.Interaction of endotoxin with particulate matter; 3.NP functionalisation for optimal endotoxin capture/scavenging in different media; 4.Synthesis of detection probes based on fluorescence total internal reflection for optimal detection and quantitative signalling of endotoxin presence. They addressed the interaction of endotoxin from different bacteria with cells, such as human monocytic cell lines, human primary immune cells, and human embryonic kidney cells transiently transfected with the known LPS receptor components and co-transfected with NFkB luciferase to receive a luminescence signal depending on LPS induced signaling. New method based on magnetic microbeads has been set up to uncover novel LPS binding molecules from promising sources such as cell lysate. Biological molecules antibodies, proteins, aptamers and anti-microbial peptides have been identified as potential new biosensors and for NP optimization/conjugation. Conjugation conditions (molecule/nanoparticle ratio, buffer, time and temperature) have been optimised in order to establish the stability of gold nanoparticles complexed with selected antibodies and proteins. Fluorescence spectroscopy method has been chosen to study the interaction between endotoxin and selected biomolecules. Total Internal Reflection Fluorescence (TIRF) configuration was established and silver nanoparticles deposited on glass surfaces has been identified to be used in the development of LPS immunoassay.
ENDONANO’s originality and innovation lies in the new ideas and technical advancement that the partners will deploy towards the development of novel unbiased methods for endotoxin detection, which could be applied to reliably measuring endotoxin also in complex matrices. The technological objectives of ENDONANO are innovative inasmuch they aim at identifying, by applying modern tools of cellular and molecular biology, material science and advanced optics, new integrated ways of endotoxin sensing and quantitative evaluation. The final innovative objective is that of translating the identified features of endotoxin detection and measurement into new knowledge for future robust and valid assays for endotoxin measurement in complex matrices. The complementarity of the expertise within the ENDONANO consortium (including industries and academic partners) will create the optimal synergy for reaching the common research training goals. The plan of fellows’ secondments will be the basis for ensuring the effectiveness of networking. The eventual research training goal, i.e. the creation of a core group of technologically advanced immuno-toxicologists, goes well beyond current programmes. ENDONANO aims at merging immunology, nanotechnology and physics into a broader approach to address toxicology, safety and related regulatory issues that is expected to attain a deeper vision of the problem, and to propose valid tools and realistic solutions.