Institute For Biomedical Engineering (IBE)

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Browsing Institute For Biomedical Engineering (IBE) by Author "Rabikoosun, Sagal"

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    Nucleic acid capture and release device.
    (Stellenbosch : Stellenbosch University, 2022-04) Rabikoosun, Sagal; Nieuwoudt, MJ; Warren, R; Grobbelaar, M; Stellenbosch University. Faculty of Engineering. Institute of Biomedical Engineering.
    ENGLISH SUMMARY: This project research aims to design a prototype device to extract nucleic acids, mainly from lysed Mycobacterium Tuberculosis cells. The proposed device would be an industry asset that provides a time-saving preparatory step for downstream genome sequencing of Mycobacterium Tuberculosis and its drug-resistant variants. Existing literature indicates that using a positive potential will electrostatically attract nucleic acids due to the negatively charged phosphate backbone in the nucleic structure. The design process is divided into phases, including planning, concept design, embodiment design, detail design, testing, and production. This project focuses on aspects of project planning to detail design; however, recommendations for testing and production are indicated. A series of experiments were developed to demonstrate that a positive potential induces a migration of nucleic acids and to display that a usable quantity of nucleic acids is released when the voltage is reversed. The testing phase includes experiments to determine the impacts of material choice, presence of a coating, the applied voltage, capture/release times, device geometry, and the influence of biological contaminants. A prototype design is proposed from these results, with an acceptance testing plan and suggestions for refinement. The quality and quantity of captured nucleic acid can be determined using several processes. In this project, to quantify changes in nucleic acid concentration, a Qubit fluorometer was used. Observations include that gold and palladium remain viable material choices; however, collection with single uncoated probes with larger surface areas works best. Nafion-coated probes collect a comparable quantity of nucleic acids regardless of the surface area, number of probes, or geometric design. The presence of a surface coating during both capture and release and a buffer during collection improved experimental repeatability. During the release, Nafion coated probes repelled nucleic acids faster than uncoated probes. However, the mean quantity of nucleic acids released is lower than that of the uncoated probe. More rigid probes are less time-consuming to surface coat and are less likely to recapture nucleic acids during the sampling process and released the most nucleic acids. In the 2-probe setup, some of the released nucleic acids are recaptured on the secondary probe once polarity is reversed and voltage lowered by 0.5 V to reduce the power output. Saturation limits were reached during the +2.5 V capture experiment at the 6 min interval on uncoated probes. All probes also reached saturation limits during the Nafion release experiment at the 1 min, 30 s interval. Nafion coatings were found to degrade in 5 % NaClO in 30 min. It is apparent that a single rigid uncoated probe of as large as reasonably possible surface area will attract the most nucleic acid in 6 min. Reversing the polarity of the probe in a buffer capture solution will gradually repel nucleic acids from the probes until an eventual saturation limit is assumed to be released, after the 2 min, 15 s interval has passed. Using a surface coating on a probe and a 2-probe setup is not advised to optimise mass captured or experimental time.