Electrochemical biosensor for detection of microRNAs in extracellular vesicles

Abstract

Extracellular vesicles (EVs) are phospholipid membrane bound sacs (vesicles) produced from almost all types of cells. They are found in circulation and contain the cargo biomolecules such as nucleic acids, proteins, lipids, and amino acids. EVs are involved in trafficking these biomolecules between cells and as such have the role in physiological and pathological processes. EVs are heterogenous and revealing their heterogeneity is crucial to understand their explicit physiological and pathological roles. Current isolation techniques cannot sort EVs based on their biogenesis and provides average information instead of each EVs subtype. Thus, single EVs analysis was popular and many surface protein characterization techniques are developed. But there are no techniques available for internal cargo analysis of individual EVs. The overall aim was to develop a technique to analyse internal microRNA cargo content, if possible, for single EVs, if not from the minimum number of EVs. To achieve that goal, light activated electrochemistry, a technique where focused light beam was illuminated on the semiconductor surface and make it electrochemically active was used. The surface was protected against oxidation during electrochemical reactions by grafting self-assembled monolayer of 1,8-nonadiyne. Then, silicon-based surface was patterned with polymers, antibodies, and cells using the light patterns. As a result, the first milestone to prepare light-assisted patterned semiconductor surface was achieved for our overall aim of analysing content of individual EVs. The size range of EVs is 30 to 200 nm, still very low compared to 30 µm which is the best spatial resolution achieved for light activated electrochemistry using crystalline silicon. Thus, chapter 4 developed a technique to improve the spatial resolution of light activated electrochemistry using amorphous silicon. Amorphous silicon has short diffusion length of charge carriers compared to crystalline silicon due to the defect states in band gap called as localized states. So, charge carriers are frequently trapped in these localized states leading to 60 times improvement in spatial resolution to 500 nm. But even this spatial resolution was not enough to analyse individual EVs. So, microRNA content from pool of EVs were detected using the screen-printed electrodes in a high throughput manner instead of single EVs.