Development of a Multi-Component Biosensor for Recombinase Polymerase Amplification-CRISPR/Cas12a-Based Detection of Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) which causes Banana Fusarium wilt is a devastating plant disease that threatens global banana production. Rapid and precise diagnostic methods is necessary for effective containment. This thesis explores the development of a multi-component biosensing platform that integrates molecular diagnostics and electrochemical detection for sensitive and portable detection of Foc TR4 DNA. The system combines microneedle-based DNA extraction, Recombinase Polymerase Amplification (RPA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a-mediated detection, and electrochemical square wave voltammetry (SWV) with targeted integration into a microfluidic system for amplification and detection within a compact heating unit. The research was organized into several key phases to achieve the thesis aim. First, a microneedle system for rapid DNA extraction of Foc TR4 was developed using polyvinyl alcohol (PVA) and tested and the initial results demonstrated successful DNA extraction from fungal mycelia. Further attempts to extract DNA from infected plant material was not successful. Following this, a custom heating system was developed to facilitate RPA DNA amplification and subsequent Cas12a-mediated cleavage. This setup ensured precise temperature control within the developed microfluidic RPA containment chip and enabled efficient electrochemical detection using gold screen-printed electrodes (SPEs) that were functionalized with methylene blue-tagged single-stranded DNA probes. To compliment this, a custom-built square wave voltammetry potentiostat was developed to quantify the electrochemical response, serving as a validation tool suitable for field applications. To validate the system performance, several complementary techniques were employed. Comparative tests with standard techniques such as the agarose electrophoresis, fluorescent detection, and naked eye visualization under LED transilluminator was used to cross-verify DNA amplification, and Cas12a activity, while a commercial Palmsens potentiostat provided benchmark electrochemical data. The multi-modal validation framework was successfully employed to detect Foc TR4. The application of the microfluidic chip with the heating system and electrochemical detection successfully demonstrated the integration of DNA amplification and Cas12amediated detection in controlled conditions. However, in the integrated platform, inconsistencies between the sensed temperature and the actual temperature within the reaction wells limited the ability to reliably perform RPA and Cas12a-mediated detection for portable real-time Foc TR4 diagnosis. Further optimization in temperature coupling, sample preparation, and system calibration is required to improve robustness. This research marks significant progress toward a portable, real-time diagnostic tool for the detection of Foc TR4 and other agricultural pathogens. The successful integration of advanced molecular diagnostics, microfluidics, and electrochemical transduction demonstrates the potential for scalable and field-ready applications. Future work will focus on addressing identified limitations, and further optimization of the system for agricultural use and enhancing system reliability across diverse environmental settings.

Grantor dc:publisher

Stellenbosch : Stellenbosch University

Year dc:date.issued

2026

Author dc:creator

• Madufor, Ndubuisi Johnkennedy

Advisors dc:contributor.advisor

• Perold, Willie J.
• Mostert, Glaudina
• Opara, U. L.

Language dc:language.iso

en

Repository record dc:identifier.uri

https://scholar.sun.ac.za/handle/10019.1/136273

OAI identifier oai:identifier

oai:scholar.sun.ac.za:10019.1/136273