Periodic Reporting for period 1 - nanoCellSense (A nanotechnology-based approach for label-free single-cell analysis of cytoplasmic proteome)
Período documentado: 2019-12-01 hasta 2021-11-30
This project aims to establish a comprehensive technique capable of in-situ, minimally invasive analysis of intracellular protein concentrations. The project hypothesises that an antibody functionalised glass nanopipette can be used to detect the presence and concentration of an antigen inside a living cell. This will be established through three main steps: 1) Functionalisation of nanopipettes by means of antibodies able to bind with high affinity the target protein; 2) Validation of the nanoprobe in cells expressing cytosolically proteins using functionalized nanopipettes; 3) Application of the nanoprobe to study cellular mechanotransduction, a hot-topic in cellular physiology and medicine research fields.
Tissues stiffen during ageing and the pathological progression of cancer, fibrosis, and cardiovascular disease. Extracellular matrix and cell stiffness increases are emerging as a prominent mechanical cue that precedes disease and drives its progress by altering cellular behaviours. Understanding the mechanisms governing cellular biomechanics and mechanotransduction may help in preventing or reversing tissue stiffening or interrupting the cellular response. The comprehension of these processes will result in novel therapeutic approach with clinical potential.
Tissues stiffen during ageing and the pathological progression of cancer, fibrosis, and cardiovascular disease. Extracellular matrix and cell stiffness increases are emerging as a prominent mechanical cue that precedes disease and drives its progress by altering cellular behaviours. Understanding the mechanisms governing cellular biomechanics and mechanotransduction may help in preventing or reversing tissue stiffening or interrupting the cellular response. The comprehension of these processes will result in novel therapeutic approach with clinical potential.
I successfully obtained nanopipettes from capillary tubes via a controlled thermal pulling process. Nanopipettes of about 80 nanometers in inner diameter centrally tapered with a half-cone angle of about 1.5°÷2° were cheaply and reproducibly fabricated. The current flowing from an Ag/AgCl electrode inserted into the nanopipette – filled with a high ionic strength solution – to a reference electrode immersed in the same bath was detected when a voltage bias is applied. The flux of ions through the nanopipette tip is sensitive to the distance between the nanopipette tip and the surface. This feature is utilised in a scanning probe microscopy technique called scanning ion conductance microscopy (SICM) to keep the nanopipette hovering above the sample surface while scanning the sample in the x-y plane.
I started exploiting a scanning ion conductance microscopy (SICM) setup mounted on top of an inverted epifluorescence microscope to acquire the three-dimensional morphology of plated cells. The same cells were characterised in their stiffness through SICM too.
In parallel to these achievements, I started working to obtain mine PC12 cells to be subsequently transfected to express a reference fluorescence cytosolic protein (GFP). These cells will be analysed through GFP-antibody functionalised nanopipettes.
I started exploiting a scanning ion conductance microscopy (SICM) setup mounted on top of an inverted epifluorescence microscope to acquire the three-dimensional morphology of plated cells. The same cells were characterised in their stiffness through SICM too.
In parallel to these achievements, I started working to obtain mine PC12 cells to be subsequently transfected to express a reference fluorescence cytosolic protein (GFP). These cells will be analysed through GFP-antibody functionalised nanopipettes.
While previous reports demonstrated the feasibility of using nanopipettes for label-free quantitative characterization of protein-protein interactions, quantitative analysis of proteins directly inside the cytoplasm of a living cell has not been achieved yet. I aim to take advantage of functionalized nanopipettes and SICM technology to accomplish this goal. Moreover, I aspect of being able to map the mechanical properties of cells, and use SICM to identify the mechanisms of mechanotransduction responsible for cell stiffening in neuronal-like cell lines. This goal will be achieved using nanopipettes to investigate the effect of mechanical forces on intracellular protein concentrations and the mechanical and morphological properties of cells.