A nanotechnology-based approach for label-free single-cell analysis of cytoplasmic proteome

ended

Presently there is a strong need to single-cell tools able to provide new insights into cellular proteome heterogeneity underlying dynamics, mechanisms and cell states in development and disease. Of particular interest in this framework is the possibility to have an effective intra-cellular proteomic detection in live cells. This mainly for three reasons: the first relates to preservation of higher protein concentration if the detection occurs before a molecule is dispersed in the extracellular environment; the second reason stems from the need to study in greater detail and in real time, single cell processes such as the molecular cell cycle or the molecular fingerprint in a label-free way; the last reason came up from the universal necessity to know the intracellular concentrations of many molecules involved in pathological and not-pathological cellular processes. I hope to achieve the desired goal taking advantage of a scanning ion conductivity microscope (SICM) endowed with a chemically functionalised ultra-sharp nanopipette to visualise living cells highlighting, from the morphological and mechanical point of view, specific target cells, that would be further investigated in their molecular fingerprint piercing the cell membrane and performing a molecular immobilisation screening. For this purpose, the inner surface of the nanopipette will be coated with a self-assembled monolayer of antibodies to target the protein of interest. The interaction between the protein and the coating of the nanopipette would result in a variation in the ionic current flowing through the pipette due to aperture occlusion. Effective binding of the protein to the inner wall of the pipette will be detected and followed in real time and, after opportune nanoprobe calibration, intracellular concentration determined. This low-perturbative, label-free approach for single cell proteome characterisation will open to new possibilities for fundamental research in cell biology and physiology.