Advances in our ability to detect biological targets relevant to human health have come from the engineering of biological molecules into assemblies capable of performing target-induced signal generation. Such assemblies, known as biosensors, are composed of a molecular recognition element (MRE) and a signal generating transduction element. One MRE class that has received great attention in recent years is functional nucleic acids, which include DNA aptamers and DNAzymes. Since 1990, a large number of functional nucleic acids have been reported. However, broad commercial use of functional nucleic acids in applications that benefit human health is sparse. The goal of this thesis is to expand the usefulness of functional nucleic acids. The thesis is made of four projects. In the first project I developed a simple colorimetric biosensor for the detection of a toxic metal ion using a reported RNA-cleaving DNAzyme coupled with urease as the signal reporter. This is followed by a project where I developed a highly effective method for the synthesis and purification of the DNA-urease conjugate needed for the biosensor. I then turned my attention to the search for high-affinity DNA aptamers that bind VEGF-165, an important human protein found to be relevant in the progression of cancers. Given that VEGF-165 is a homodimeric protein, in my third project I looked into the suitability of reported DNA aptamers for this protein for the creation of dimeric aptamers with higher binding affinity. I examined multiple factors that may affect the successful engineering of dimeric aptamers and determined that none of the existing aptamers are compatible for creating a productive dimeric aptamer. With this finding, I made an effort to create our own aptamers for this protein target. I was able to isolate a new aptamer that appears to be an excellent candidate for creating a higher affinity DNA aptamer. Overall, my work adds to our increasing appreciation of the functional capability demonstrated by single-stranded DNA molecules. More importantly, I hope the methods I have developed and new functional DNA molecules I have generated in this thesis will continue to drive the development of the functional nucleic acid field and contribute to the health research community’s efforts to increase human longevity. / Thesis / Doctor of Philosophy (PhD)

The reaction between 2 acetamino 6 amino benzo thiazole and chloroiridio acid is not sensitive enough to be of importance as a colorimetric quantitative reaction. IrCl₆-- can be detected with this organic compound if iridium is present in as much as 0.02 mg. per ml. of solution providing ferric iron is absent. If ruthenium is present it must be removed before the test for the iridium anion can be made. / M.S.

M.S.

Cytomegalovirus (CMV) is a member of the Herpesviridae family and is known to infect people of all ages. In most cases, CMV infection is asymptomatic, and the virus is cleared from the host without showing any significant symptoms. However, 1 out of 200 babies are born with congenital CMV infection, which affects multiple organs, including the brain, liver, spleen, lung, and inner ear. One long-term health problem in 1 out 5 babies born with congenital CMV infection is hearing loss. The progression of CMV-associated hearing loss in the first two years of life may lead to developmental delays in language, learning, and communication. Currently, for serological testing of CMV in patients older than 12 months, real-time polymerase chain reaction (rtPCR) is used. Although the rtPCR method quantitatively detects the CMV, this method is expensive and needs highly skilled technicians to perform the assay. Therefore, there is a need for a cost-effective, simple, and rapid diagnostic tool that can help detect CMV in newborn babies and prevent CMV-mediated pathology in pediatric as well as in future adult conditions. In recent scientific studies, gold nanoparticles (AuNP) of 100 nm, 40 nm, and 15 nm sizes have shown promising results in detecting various viruses. In our study, we developed an AuNP-based colorimetric assay for detecting CMV in pediatric-derived urine samples. For comparison purposes, the pediatric urine samples were screened through quantitative PCR (qPCR) for positive and negative CMV determination. In our assay, we explored the ability of different-sized nanoparticles to detect CMV in pediatric urine samples. Using purified CMV virions as a positive control, we have shown that AuNP can effectively detect the presence of CMV in pediatric urine samples. Therefore, this colorimetric assay may be a basis for a useful diagnostic tool for detecting CMV in newborn babies. It is a rapid and non-invasive high-throughput assay for screening for the presence of CMV at the point of care and may help provide better therapeutic outcomes for pediatric patients.

Call number: LD2668 .T4 1984 T78 / Master of Science

Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this dissertation, we demonstrate that chromophores such as permanganate and bicinchoninic acid (BCA) and copper, however, when combined with nucleobase-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of cytosines, adenines and/or guanines in single-stranded DNA oligomers, even in the presence of thymines. Furthermore, the reactions are stoichiometric, which allows for the quantification of cytosine, adenine and/or guanine frequency in these oligomers. The BCA/copper reagent detects the reducing sugar, 2-deoxyribose, resulting from the chemical cleavage of a given nucleotide’s N-glycosidic bond. Therefore, these colorimetric assays are effectively detecting abasic sites in DNA oligomers, which are known to occur in damaged DNA. Our analytic approach termed colorimetric identification of exposed nucleic acids (CIENA) combines the use of BCA/copper, permanganate, and diphenylamine chromophores along with digital image capture to identify and quantify each nucleobase within DNA. The digital image color properties are quantified in terms of the image’s hue, saturation, and lightness using the CIELAB color space and ΔE quantification of color. CIENA is a simple, low-cost tool that could be applicable in various types of nucleic acid analyses, such as the quantification of nucleobase composition and the identification of damaged DNA.

Sol-gel porous glasses have been doped with indicator molecules for colorimetric measurements of pH and metal ions in solution. pH measurements were made in real time (20 seconds) to a wide pH range 3-8 by using bromophenol blue doped in sol-gel thin films. New methods of sol-gel coating on the inside of test tubes and tubing have been introduced for simple, non invasive, on the spot chemical sensing. pH indicator doped films were also successfully autoclaved for biological applications without affecting their chemical and physical properties. The pore structure of thin films has been controlled for minimising the effect of ageing on response time by introducing dimethyl formamide (DMF). The effect of light, temperature and salt on thin films have been studied. The results show that they are relatively stable between 20-31' C and less affected « 0.03 absorbance unit decrease in 3 weeks) by light. However their response to pH is changed by adding salt in solution with concentration higher than O.OlM. Fourier transform infra red (FTIR) study of films has been conducted to elucidate the effect of ageing, DMF and autoclaving on their chemical structures. It was found that ageing continues after four weeks of fabrication and addition of DMF helps to reduce ageing and increase porosity. The long term stability of these pH indicator doped films in various solvents has been established. Thin films on microscope slides were deposited by using a newly designed spin coater and have been demonstrated as reusable pH slides. Sol-gel films were also doped by different metal reagents. Eriochrome cyanine doped thin films were found to be sensitive to copper ions in solution. Copper (Cu++) was measured to a low concentration of 0.6 ppm. The effect of light and temperature on Eriochrome copper complex was studied. Interferences of other metal ions were examined. A fibre-optic pH sensor has been demon.strated by coating an optical fibre with a sol-gel film (0.8 J.1m thick) doped with bromophenol blue. The sensor has shown fast response (5 seconds) to pH changes from pH 3 to 8 and no leaching or cracking during repeated use. It is simple to fabricate and easy to use as an interchangeable pH fibre probe. It has potential application in biological processes as an integral part of an online monitoring system.

This research focused on the cytotoxic properties of RRR-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) in transformed and cancerous cell lines. We used RAW264.7 macrophage and prostate cancer (LNCaP) cell lines in this study. TPGS caused cell death and decreased cell viability in a dose and time dependent manner. Cell death was evaluated fluorimetrically by employing the nucleic acid-binding fluorophore; propidium iodide. A colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. Cell death can occur through necrosis or apoptosis. Our results suggested that TPGS triggered apoptotic cell death. Induction of apoptosis, as measured by caspase 3 enzymatic activity, was dependent upon the TPGS dose and incubation time. Caspase 8 was activated before caspase 9, suggesting the importance of the death receptor pathway in apoptosis. Our results indicated that TPGS cytotoxicity could also be due to one of its products of hydrolysis, alpha-tocopheryl succinate.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Point-of-care testing (POCT) devices have received increasing attention because of their potential to address the urgent need for quick and accurate diagnostic tools, especially in areas of personal care and clinical medicine. They offer several benefits over current diagnostic systems, including rapid diagnostic results in comparison to microbial cultures, simple interpretation of results, portability, and requiring no specialised laboratory equipment or technical training to operate. These are essential for diagnosing critical illnesses, such as sepsis, in areas of poor healthcare infrastructure. Sepsis, an innate physiological response to infection, is a growing problem worldwide with high associated costs and mortality rates, and affects a wide range of patients including neonates, infants, the elderly, and immunocompromised individuals. A literature review of the biomarkers of sepsis and the currently available diagnostic systems indicates the need for a biosensor capable of meeting the requirements of designing POCT systems and achieving detection of low concentrations of biomarkers. To meet these demands, two significant contributions to developing POCT platforms have been achieved and described in this thesis, including: 1) development of a colorimetric, magnetically separable biosensor that can be easily fabricated and demonstrates an easily identifiable colour response upon analyte detection, as well as the ability to capture and detection target biomarkers at low concentrations from complex solutions; and 2) tuning of the biosensor’s colorimetric response to achieve low detection limits, as well as demonstration of the versatility of the biosensor for sensing different target analytes. The developed biosensor in this work combines colour responsive polydiacetylenes and superparamagnetic iron oxide for the first time to achieve a biosensor capable of meeting these demands. The sensors exhibit identifiable colour responses to biomolecule detection, capture of a target analyte from complex solutions, sensing of different target analytes, a lower detection limit of 0.01 mg/mL, and rapid separation from solution with a common magnet. This work has been a significant demonstration of the capabilities of this biosensor as a new platform for POCT systems to diagnosis sepsis, and potentially other sensing applications.