DOCTORAL CANDIDATE 6
Mid-IR near-field imaging (AFM-IR) of human milk extracellular vesicles
IIS La Fe
Location: Valencia, Spain
Supervisor: Dr. Julia Kuligowski
Technische Universität Wien
Location: Vienna, Austria
Supervisor: Dr. Georg Ramer
OBJECTIVES
At IISLAFE, the DC will optimize the collection of human milk (HM) and subsequent isolation of HM extracellular vesicles (EVs) using Size Exclusion Chromatography and ultracentrifugation. Isolated EVs will undergo comprehensive characterization, including size, shape, protein content, lipid analysis, and cargo assessment. This workflow will be applied to HM samples from mothers of infants with and without NEC. At TU-WIEN an AFM-IR set-up (Bruker nanoIR1) will be adapted for the study of HM EVs. The standard ZnSe ATR carriers will be replaced by novel Si-ATR elements from IRUBIS and metasurfaces (DC10,11). This will allow to immobilize antibodies, such as anti-CD9 antibodies which bind to the CD9 antigens present in the EVs, to selectively retain HM EVs on the surface. After rinsing the prepared samples are ready for measurement by AFM-IR (with DC4). Finally, to increase selectivity toward selected proteins an existing photothermal IR setup at TU Wien will be upgraded by adding up to four probe lasers for detection of multiple temperature sensitive fluorophores after photothermal excitation. DC6 will be fully trained on various analytical chemistry assays and industrial downstream processing along with key spectroscopy skills. DC6 will work with DC1 and DC4 to define target specification for the lights sources and provide feedback on use.
EXPECTED RESULTS
With the high spatial resolution of AFM-IR there is a high chance that it will be possible to measure individual HM EVs. Based on the information content in the AFM-IR spectra and by using the bulk reference analytics we are optimistic to provide detailed information on the chemical composition of HM-EVs of different size. Furthermore, we expect to be able to study variations in the composition of individual HM EVs depending on the gestational/postnatal age of the infants. We expect gaining insight into the connection between maternal factors and the biochemical composition of HM EV subpopulations as well as the interplay between the composition of HM EV sub-populations and infant microbiota, inflammatory markers, and oxidative stress biomarkers. The results will be discussed with medical doctors at IISLAFE.
Planned Secondment:
InsuCaps Ltd
Ph.D. Awards From:
Universitat de València
Technische Universität Wien
N.B. Secondments are indicative and may be subject to change
This project is part of Work Package 2
for Work Package 2 Overview