Microfluidics have been created to acquire, operate, and process complex fluids in extremely tiny volumes with high efficiency and high speed, and without the requirement for an experienced operator. In addition, microfluidic systems also enable miniaturization and incorporation of different complex functions, which can help bring intricate diagnostic tools out of the laboratories. Ideally, these systems should be inexpensive, precise, reliable, robust, and well-suited to the medical diagnostic systems. Most of the microfluidic devices reported previously were based on devices made of polydimethylsiloxane (PDMS). PDMS is a material that dissolves in many common organic solvents. Meanwhile, it is also prone to absorb small molecules like the proteins, which is detrimental to a stable and reliable result. Current work focuses on bioassays that are badly needed in our life and these bioassays are addressed based on microfluidic platform with different materials. The translation of microfluidic technology into large scale implementations highly relies on new materials that address the limitations of PDMS. Firstly, we fabricated two different microfluidic platforms for rapid antimicrobial susceptibility testing (AST). One was made of hydrogel, and the bacterial cells were cultured on the top of the device; the other was of polypropylene (PP), and bacterial cells were cultured inside the microchannels. Meanwhile, we developed a novel "barcode" sensor, a microscope-free method for cell accumulation and cell counting, as the downstream of the PP-based chips. As a result, AST can be accomplished simply through an application on a mobile phone rather than using an expensive and sophisticated microscope. Secondly, we presented a self-contained paper-based system for lead(II) ion detection based on G-quadruplex-based luminescence switch-on assay, comprising a novel type of paper-based chip and a matching portable device. Different from the reported paper-based devices, the paper substrate we chose was art paper, which is used for printing magazines. This type of paper could prevent the absorption of liquid into the paper matrix and hold the liquid in place for a period of time; and it could also be used for temporary liquid containing like a plastic substrate (such as polypropylene (PP) and polystyrene (PS)), but the surface of the paper is inherently hydrophilic. In such a design, liquid drops are suspended on the surface of the device in designed reservoirs, rather than absorbed into the paper; when the chip is tilted, the liquid drops will move to other reservoirs according to the guidance of channels defined on the surface. To differentiate it from reported μPAD devices that are fabricated with water-permeable paper, we name this new type of paper-based devices suspending-droplet mode paper-based microfluidic devices (SD-μPAD). Different from the conventional μPADs that use capillary force to drive liquid, our SD-μPADs uses wetting and gravity as driving force. To fabricate the superhydrophobic pattern on the paper device, we developed a new microcontact printing-based method to produce inexpensive and precisely patterned superhydrophobic coating on paper. The coating material is poly(dimethylsiloxane) (PDMS), a hydrophobic and transparent silicone that has long been used for fabricating microfluidic devices. Importantly, the negative-relief stamp we used is made of Teflon, a non-stick polymer, so that the PDMS-coated paper could be peeled from the stamp flawlessly. After such fabrication process, the stamped area of the paper is coated with a textured PDMS layer that is decorated with arrays of micropillars, which could provide superhydrophobic effect and most effectively hold the droplets in place; the remaining area of the paper is still hydrophilic. As a demonstration of this new design, we developed a method using the reaction characteristics of iridium(III) complex for rapid, onsite detection of lead(II) ions in liquid samples. As the reagents have already been loaded onto the paper device during fabrication, the only reagent the users need to add is water. Because of the large Stokes shift of the iridium(III) complex probe, inexpensive optical filters can be employed, and we were able to make an inexpensive, battery-powered compact device for routine portable detection using a smartphone as a detector, allowing the rapid analysis and interpretation of results on site as well as the automatic dissemination of data to professional institutes, including tests even in poor rural areas in developing countries. Thirdly, we upgraded our suspending-droplet mode paper-based microfluidic device (SD-μPAD), which is used for the detection of lead(II) ions in liquid solution. The reason is that our paper-based SD chips are not suitable for long reaction process (> 20 min) detection of biomolecules due to the potential permeation and contaminating problems of art papers. Hence, we chose polypropylene (PP), a hydrophobic, cheap, and thermal stable material (< 110°C), as the material for the fabrication of the SD microfluidic chip. We established a convenient, low-cost, portable and reliable platform for monitoring VEGF165 accurately, which can be applied for point-of-care (POC) testing. In this project, we also employed the label-free oligonucleotide-based luminescence switch-on assay on the microfluidic platform, which possesses the advantages of high sensitivity and high selectivity. Based on the detection of VEGF165 in a three-step reaction process, we adopted a new design for the droplet transfer throughout the channels. This design could migrate the droplet through the chambers via controlling the orientation of the chip, which systematically combined the superhydrophobic force of the coating, the gravity of the droplet and the surface tension between PP and droplet. Therefore, traditional micro pump could be avoided and the total cost for the device could be substantially reduced. In addition, we developed an automatic, matched and portable device for the detection of VEGF165, which assembled by a rotatable chip holder, a UV lamp, a filter, and a camera. Finally, we developed a new whole Teflon membrane-based chip for the aptamer screening. Our article "Whole-Teflon microfluidic chips" introduced the fabrication of a microfluidic device entirely using Teflon materials, one group of the most inert materials in the world. It was a successful and representative introduction of new materials into the fabrication of microfluidic devices, which show dramatically greater anti-fouling performance. However, even such device was inadequate for current purpose, as it is rigid and lacks convenient valve control functions for particle suspensions used in systematic evolution of ligands by exponential enrichment (SELEX). For this project, we propose a SMART screening strategy based on a highly integrated microfluidic chip. This new type of whole-Teflon devices, which are made of flexible Teflon membranes, offering convenient valving control for the whole SELEX process to be performed on chip and fulfilling the anti-fouling requirement in the meantime. The SELEX cycles including positive and negative selections could be automatically performed inside tiny-size microchambers on a microchip, and the enrichment is real-time monitored. The selection cycles would be ended after the resulted signal of the aptamers with high specificity reached a plateau, or no target aptamer is captured after a number of cycles of enrichment. Owning to the antifouling property of the chip materials, the loss of the sample is tremendously reduced. The SMART platform therefore is not only free of complicated manual operations, but also high-yield and well reproducible over conventional methods

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

An extract of the tuber of Callilepis laureola is regarded as the source of a powerful therapeutic agent, known as Impila. Its use is associated with fatal hepatic and renal necrosis, the renal toxin being atractyloside (ATR). The aims of this study were threefold. Firstly, to generate a model set of biological specimens (urine, serum, liver and kidney) from rats dosed with 5-25 mg ATR/kg bwt. Secondly, to develop a competitive ELISA and HPLC method for the diagnosis of ATR poisoning employing the model specimens as test samples. Thirdly, to localise the target organs, cells and organelles of ATR, in vivo. The HPLC method necessitated the systematic development of the derivatisation of ATR with 9-anthryldiazomethane, sample clean up employing hexane, methanolic hydrochloric acid and a silica minicolumn, as well as the chromatographic conditions. Optimal resolution was obtained with a 3.9 x 150 mm NovaPak reverse phase column, fluorescence detection (excitation = 365 nm, emission = 425 nm) and a solvent system of MeOH:1M ammonium acetate:1M glacial acetic acid:water (38:2:2:58). This method has a detection limit of 0.001 ng ATR, shows a mean recovery of 89% and detected approximately 6.7 ug ATR/g wet weight of tuber tissue. The toxin was also detected in some of the urine samples at levels of about 200 pg/ml, but not in the serum. The production of antibodies to ATR for use in the ELISA and immunocytochemical investigations required the investigation of the conjugation procedure, carrier type, host species and immunization protocol. Optimal antibody yield, specificity and affinity was obtained with an acid-treated Salmonella minnesota bacterial carrier conjugated to ATR by carbodiimide, although there were indications of class switch inhibition and Tlymphocyte suppression by ATR. The development of the ELISA yielded a protocol involving the coating with a bovine serum albumin-ATR conjugate, blocking with bovine serum albumin, incubating the primary antibody at 4°C and detection with a secondary antibody-alkaline phosphate conjugate. This method detected ATR in both urine and serum from ATR-dosed rats and shows a detection limit of 10 ng. Since the less sensitive ELISA detected ATR in samples where the HPLC did not, this suggested that ATR is biotransformed in vivo, such that its retention time on a reverse phase column is affected, but not its epitope determinants. The results of the organ function assays demonstrated that, when administered intra-peritoneally, ATR is not hepatotoxic, but is a powerful nephrotoxin, targeting for the microvilli of the brush border of the proximal tubules, and compromising glomerular permselectivity and distal tubular function. In addition, this toxin inhibits proline transport in the proximal tubule, and therefore probably affects protein biosynthesis. Renal regeneration is noted 3 days post-dosing, as demonstrated by calcium excretion. Immunocytochemistry was optimised on tuber tissue and necessitated the intracellular fixation of the toxin, using carbodiimide, to prevent leaching out of the ATR. The toxin was encapsulated in vesicles in the tuber tissue. Atractyloside was also located in the kidney of ATR-treated rats, up to 72 hours after exposure, targeting the microvilli of the proximal tubule brush border, the mitochondrial cristae and specific sites on the Golgi apparatus membrane. Microvilli disruption and mitochondrial swelling was noted within 24 hours after exposure to the toxin while after 72 hours, loss of mitochondrial integrity was observed. The development of these diagnostic assays for ATR have provided the means to monitor the levels of this toxin in plant extracts and mammalian body fluids. Future work should include the identification of the hepatotoxin associated with Impila, the effects of the route of administration on the toxicity of this remedy and furthermore, the identification of a suitable antidote, which could include the use of duramycin and stevioside. The association between compounds blocking the ADP/ATP antiporter in the c-state and Reye's syndrome should also provide an interesting area of research. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1991.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 129-137).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 294-317).

Thesis (M.D.) - University of St Andrews, November 2008. / Restricted until 12th November 2010.

The purpose of the present study was to compare the amounts of vitamin A in sweet potato flour with that of carrot flour and dehydrated carrots by using the biological assay method.

A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.

Submitted in partial fulfillment of the requirements for the degree of Doctor In Technology (Clinical Technology), Durban University of Technology, Durban, South Africa, 2016. / Erythropoietin (EPO) is widely used in patients with chronic renal failure and is a necessity. However, due to the cost implications and the medical complications in our population it is imperative to review the factors affecting the process of erythropoiesis and the analysis of cell proliferation and cell viability in the bioassay. Complications such as hypertension and risk of worsening a malignancy cannot be ignored. We had previously analysed variations of erythropoietin levels in haemodialysis patients over a six month period. This study aims to evaluate erythropoiesis in conjunction with various laboratory, demographic, clinical parameters and inflammatory markers, in the population of haemodialysis patients. EPO, antibody level and antibody activity were analysed in the population groups as EPO responsive and EPO sensitive patients. This is a prospective, experimental and controlled study. Fifty nine patients were randomly selected from haemodialysis units of Addington and King Edward VIII Hospitals following an informed consent and 15 healthy individuals were also selected as controls. Demographic parameters (age, sex), clinical parameters (weight, height, skin folding, EPO doses and blood pressures (BP) were recorded. Pre-dialysis serum was used to measure laboratory markers (haemoglobin, transferrin, ferritin, albumin, ESR, C reactive protein, creatinine and urea). EPO levels and antibody levels were measured by ELISA, the optical density of each well was determined within fifteen minutes using the microplate reader set at 450 nm. All results were statistically analysed using SPSS statistical package version 21 (IBMR). Patients requiring very high doses of EPO to reach Hb of 11g/dL, and they remained anaemic after at least three months of adequate EPO doses were considered to be EPO resistant. Those who responded to the usual EPO doses were labelled EPO sensitive. The bioassay was used to quantify cell proliferation and cell viability in the presence of EPO. The UT 7 cells were cultured in medium, in the presence of serum from the EPO resistant, EPO sensitive patients and the healthy, control subjects. Luminescence was read with the Glorunner Microplate Luminometer and was recorded in relative light units (RLU). The analysis revealed: a non-significant positive correlation between haemoglobin and erythropoietin levels. However, a strong negative correlation was found between CRP and albumin level (R= -0.591; (p=0.001), which was not significant. No correlation was found between haemoglobin or erythropoietin levels and CRP or albumin. There was a positive correlation with systolic and diastolic blood pressures and mean arterial pressures which was statistically significant (p <0.05). EPO dosages and Hb levels were correlated significantly (p < 0.05). No correlation of EPO levels and Hb; age and Hb was found to be significant (p = 0.08). The UT 7 cells cultured in serum in medium alone with RHuEPO containing cells were statistically significant (p <0.01)). Reduction of ATP stimulation between medium and serum was observed. However, mean arterial pressures had a significant association with EPO resistance (p = 0.041) odd ratio- 1.066. In conclusion, EPO level is not a useful tool for the monitoring of its use as it does not correlate with EPO goal of red blood production in our patients. The neutralizing antibodies did not correlate with any of our variables contributing to erythropoiesis, and are therefore not confirmed as playing a major role in erythropoiesis. From the analysis of our results the key contributing factors of EPO doses, malnutrition and age were more significant in erythropoiesis. However the higher doses of EPO significantly increased the blood pressures and the mean arterial pressures (MAP). The analysis of the bioassay showed lack of difference between EPO responsive and EPO sensitive patients. This observation warrants further studies to clarify the role of serum of haemodialysis patients in erythropoiesis. / D

A genetically determined high level of intrinsic normal tissue radiosensitivity may account for the 5% of patients who experience unexpectedly severe normal tissue side effects following radiotherapy. The pre-treatment identification of these individuals by a diagnostic test or “predictive assay “ may allow appropriate modification of treatment plans and improve the therapeutic index of radiotherapy. Results from studies of cell-based assays measuring the response of a single cell type taken from patients to in vitro irradiation have been inconsistent, leading to the opinion of many that they are of no value in the prediction of normal tissue radiosensitivity. A systematic review of the literature presented here, however, suggests that poor methodology of study design often with inadequate control for those factors other than normal tissue radiosensitivity which influence radiotherapy toxicity and lack of reporting of assay precision means that it is difficult to form any conclusions, positive or negative about the diagnostic accuracy of the cell-based assays studied so far. Analysis of individual patient data extracted from these studies suggests that at least some of these assays may possess some discriminatory value. This finding justified an attempt to develop a novel cell-based assay based on the kinetics of radiation-induced .H2AX in peripheral blood lymphocytes. Assay failure rate was high and intra- and inter-sample assay reproducibility was poor for quantification by microscopy but were better for flow cytometric analysis. A study of 8 volunteers, however, demonstrated that intra-individual variation was higher than inter-individual variation in assay results, strongly suggesting that poor assay reproducibility due to technical or biological factors may limit the assay’s potential to identify radiosensitive individuals. This suspicion needs to be confirmed in a clinical study of patients of known radiosensitivity. As blood sample storage conditions affect assay results these will need to be standardized to prevent confounding of results.