Browsing by Author "De Jager, Ruan"
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- ItemDesign and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux(Stellenbosch : Stellenbosch University, 2021-03) De Jager, Ruan; Perold, W. J.; Loos, Ben; Stellenbosch University. Faculty of Engineering. Dept. of Electronic and electrical engineering.ENGLISH ABSTRACT: n 2016 professor Yoshinori Oshumi proved that autophagy is dysfunctional in neu- rodegenerative diseases such as Alzheimer’s, enhanced in various cancers and progressively declining in ageing. Autophagy also relates to metabolic disorder, infectious and general degenerative diseases, and is a critical factor in the development of diseases relating to an increase in cancer risk. The ability to monitor autophagy has become key interest in the bio-tech industry and is also an excellent indication of a person’s overall health, however to monitor and determine autophagy flux remains a challenge. LC3 protein is a proven biomarker that directly relates to the rate of autophagic ac- tivity and accumulates as autophagosomes are formed. With an increase and decrease in the rate of autophagy the concentration of LC3 levels will alternate and is an excellent estimate towards determining autophagy flux. For this project different biosensing techniques were explored for LC3 detection with an electrochemical biosensor chosen to be most applicable due to its robustness and se- lectivity towards proteins. The goal towards a proof of concept utilised commercially manufactured carbon nanofibre screen printed electrodes by Dropsens that were modified in development of an immunosensor. For antibody crosslinking, the sensors were electro- grafted using a diazonium salt mixture to form carboxyl support groups followed by the activation of these groups using EDC/NHS activation chemistry. The antibody bindings were verified with fluorescent microscopy by attaching a secondary antibody. Square wave voltammetry was use as method of detection due to its simplicity as well as the method generating absolute data for comparison. All square wave voltammetry experiments were executed in 5 mM ferricyanide redox couple. The functionality of the sensors were tested by performing a full scale test over a wide range of different protein concentrations. The work done was successful towards a proof of concept sensor for LC3 detection, with an increase in protein bindings observed over the tested range and space for optimisation to increase sensitivity and accuracy.