Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux
Thesis (MEng)--Stellenbosch University, 2021.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2021
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613792125321216 |
|---|---|
| access_status_str | Open Access |
| author | De Jager, Ruan |
| author2 | Perold, W. J. |
| author_browse | De Jager, Ruan Perold, W. J. |
| author_facet | Perold, W. J. De Jager, Ruan |
| author_sort | De Jager, Ruan |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MEng)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/110490 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:46.341Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/110490 Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux De Jager, Ruan Perold, W. J. Loos, Ben Stellenbosch University. Faculty of Engineering. Dept. of Electronic and electrical engineering. Immunosensor -- Design and Fabrication UCTD Degenerative diseases Cancer -- Risk factors Autophagy Flux Biosensors Thesis (MEng)--Stellenbosch University, 2021. 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. AFRIKAANSE OPSOMMING: In 2016 het professor Yoshinori Oshumi bewys dat autofagie disfunksioneel in neuro- degeneratiewe siektes is soos byvoorbeeld Alzheimer’s, versterk is in ’n verskeindenheid kankers en geleidelik afnemend in veroudering is. Autofagie het ook verband met metabo- liese agteruitgang en aansteeklik asook algehele degeneratiewe siektes, en speel ’n kritiese rol in die ontwikkeling van siektes wat in verband met kanker risiko is. Die bepaling en monitering van autofagie se aanvraag het vermeerder maar bly tans ’n groot uitdaging. LC3 proteïn is ’n bewysde biomerker vir autofagie en staan in direkte verband met autofagie vloed waar die versameling van die proteïn verwys na die ontwikkeling van ’n outofagosome. ’n Vermeerdering en vermindering in autofagie vloed sal ’n verhoging en verlaaging in LC3 voortbring. ’n Elektrochemiese biosensor was mees van toepasing op gevolg van die literatuur stu- die omdat dit robuust en selektief op proteïne is. Die hoof konsep begrip is bewys deur gebruik te maak van kommersiele vervaardigde koolstof nanovesel elektrodes en dit deur prosesse te plaas namens die ontwikkeling van ’n immunosensor. Vir teenligaampie kruis koppeling was die sensors ge-electrograft met ’n diazonium sout mengsel om karboksiele groepe te vorm en is geaktiveer deur gebruik te maak van EDC/NHS kruis koppeling chemie. Fluoresserende mikroskopie was gebruik om die bindings te verifieer deur ’n sekondˆere teenligaampie te bind. Blok golf voltammetrie was gebruik as die hoof metings metode en is uitgevoer in 5 mM ferricyanide. Die sensor se funksionaliteit was getoets deur ’n volskaalse toets uit te voer en sy werking oor a wye spektrum van konsentrasies se evalueer. Die ontwikkeling van ’n bewys van konsep begrip was suksesvol waar ’n vermeerdering in konsentrasie waargeneem is op die geproduseerde data, met baie spasie vir optimeering om die sensor se sensitiwiteit en akuraatheid te verbeter. Masters 2021-05-24T11:42:38Z 2021-05-24T11:42:38Z 2021-03 Thesis http://hdl.handle.net/10019.1/110490 en_ZA Stellenbosch University 127 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Immunosensor -- Design and Fabrication UCTD Degenerative diseases Cancer -- Risk factors Autophagy Flux Biosensors De Jager, Ruan Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title | Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title_full | Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title_fullStr | Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title_full_unstemmed | Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title_short | Design and fabrication of a label-free LC3 immunosensor towards the estimation of autophagy flux |
| title_sort | design and fabrication of a label free lc3 immunosensor towards the estimation of autophagy flux |
| topic | Immunosensor -- Design and Fabrication UCTD Degenerative diseases Cancer -- Risk factors Autophagy Flux Biosensors |
| url | http://hdl.handle.net/10019.1/110490 |
| work_keys_str_mv | AT dejagerruan designandfabricationofalabelfreelc3immunosensortowardstheestimationofautophagyflux |