The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.

Magister Scientiae - MSc / Cancer, the second most common cause of death after heart disease, is a complex and multifactorial disease that up to date is still under extensive research. To achieve early detection of cancer disease the discovery of specific, sensitive and reliable biomarkers is required. Telomerase is a ribonucleo-protein complex that has been identified as an important target for cancer diagnostics and cancer therapy, because 85% of more than 950 primary tumours express telomerase activity. The standard method for the detection and quantification of telomerase activity is the polymerase chain reaction (PCR)-based assay known as the telomeric repeat amplification protocol (TRAP) assay. TRAP and other methods developed for telomerase detection have limitations for example its time consuming, requires complicated machinery, expensive equipment and reagents thus there is a need for a more sensitive, reliable and high-throughput method. Electrochemical biosensors are quickly emerging as an alternative for early detection of cancer because they can be designed to detect developing cancer biomarkers and to allow improved monitoring of cancer growth and patient therapy. This research study reported for the first time the successful fabrication and implementation of highly sensitive 3-mercaptopropionic acid indium telluride quantum dots (3MPA-In2Te3 QDs) based genosensor for detection of telomerase biomarker. The colloidal poly-dispersed 3MPA-In2Te3 QDs introduced into the genosensor system were successfully synthesized by a simple, inexpensive and reproducible aqueous method. The as prepared 3MPA-In2Te3 QDs was characterized by Ultraviolet Visible (UV-VIS) spectroscopy, Fluorescence (FL) spectroscopy, X-ray Diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy and High Resolution Transmission/Scanning Electron Microscopy (HR TEM/SEM). Electro-analysis of 3MPA-In2Te3 QDs was done by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). HR-TEM studies revealed formation of small sized QDs about 6 nm in diameter while UV-VIS studies showed presence of iv absorption peaks in the ultraviolet region (100-400 nm) which confirmed the formation of these small sized QDs. The good electrochemical, optical, physical and chemical properties of the 3MPA-In2Te3 QDs allowed them to be used as a mediating platform between deoxyribonucleic acid (DNA) and gold electrode (AuE). The successful detection of telomerase was achieved by hybridization process between the probe single stranded deoxyribonucleic acid (ssDNA) drop coated on the 3MPA-In2Te3 QDs/AuE surface and its complementary ssDNA in biological buffer solution (0.10 M tris-ethylenediamine tetraacetic acid (TE) buffer solution, pH 8.00). The response of the 3MPA-In2Te3 QDs based genosensor towards different concentration of complementary ssDNA was studied by CV, square wave voltammetry (SWV) and EIS. It was observed that all three analytical techniques exhibited good linearity since their linear correlation coefficients (R2) corresponded to 0.99. However, it was observed that EIS was the best technique for the detection of telomerase compared to both CV and SWV since it showed a higher sensitivity (2.44 Ω/nM) towards detecting telomerase with a detection limit as low as 0.00014 ng/mL. Control experiments were also carried out by monitoring the hybridization process in the presence and absence of complementary ssDNA and it was determined that the QDs based genosensor was highly selective towards complementary ssDNA. In view of the attractive analytical characteristics and advantages, the ultimate goal of the developed QDs based genosensor is to apply it in real clinical samples of cancer cells or bodily fluids of cancer patients for the detection of telomerase cancer biomarker.

Abstract Biopharmaceuticals are growing in medical and economical significance, replacing several chemically synthesized pharmaceuticals and providing new treatments for serious diseases. Research within the biopharmaceutical field is expensive and labor intensive with the majority of work featuring diverse bioprocesses. Miniaturization of bioprocesses into microbioreactors, miniaturized bioprocess vessels for diverse experiments, reduces the amount of necessary reagents as well as time and labor due to the possibility of multiplexing. A crucial step of research is analytics, commonly performed by expensive and time consuming standard laboratory based analytical tools that require larger sample volumes that than microbioreactors can provide. Biosensors are analytical devices that can provide highly sensitive and selective online detection (in situ) and lend themselves to miniaturization. Biosensors detecting glucose, lactate, pyruvate and galactose were developed and investigated herein. 3D printing and laser ablation were used as fabrication techniques, enabling development of unit operations (mixing and pumping) necessary for biosensor integration into microbioreactors. A 3D printed microbioreactor was developed with integrated glucose and optical density sensors for the cultivation of yeast where the biosensor was found to provide critical online data of glucose concentration, thus enabling bioprocess control. Additionally, molecularly imprinted polymers were investigated as novel recognition components due to their increased stability compared to biological molecules within bioprocess environments. A molecular imprinted polymer for folic acid was developed and tested as a sorbent in a microfluidic solid phase extraction system for detection. / Tiivistelmä Biofarmaseuttisten valmisteiden lääketieteellinen ja taloudellinen merkitys kasvavat. Valmisteet korvaavat kemiallisesti valmistettuja yhdisteitä ja tarjoavat uusia hoitoja vakaviin sairauksiin. Biofarmaseuttinen tutkimus on kallista, ja erityisesti bioprosessien kehittäminen vaatii paljon työtä. Bioprosessien miniatyrisointi mikrobioreaktoreita käyttäen vähentää tutkimuksessa tarvittavaa reagenssien kulutusta, lyhentää aikaa ja pienentää työmäärää, koska mikrobioreaktoreiden avulla voidaan tehdä useita rinnakkaisia kokeita samanaikaisesti. Keskeinen osa bioprosessien tutkimusta on niiden seuraamiseen tarvittava analytiikka. Yleensä seuranta tehdään kalliilla ja aikaa vievillä analyyttisen kemian menetelmillä, jotka vaativat suurempia näytetilavuuksia kuin mitä mikrobioreaktoreista saadaan. Biosensorit ovat erittäin herkkiä ja mittauskohteen tarkasti tunnistavia analytiikkavälineitä, joilla voidaan saada online-mittaustietoa suoraan mittauskohteessa. Ne pystytään myös miniatyrisoimaan. Tässä tutkimuksessa kehitettiin aluksi glukoosin, laktaatin, pyruvaatin ja galaktoosin biosensorimittaukset. Sensorilaitteiden valmistustekniikkoina käytettiin 3D-tulostusta ja laserleikkausta. Näin pystyttiin kehittämään yksiköt myös näytteen sekoittamiseen ja pumppaamiseen. Lopuksi hiivasolujen kasvatusta varten valmistettiin 3D-tulostettu mikrobioreaktori, johon yhdistettiin glukoosisensori ja optisen tiheyden mittaus. Glukoosin mittaus tuotti glukoosin pitoisuudesta onlinetietoa, mikä on erittäin tärkeää prosessin valvonnalle ja ohjaukselle. Lisäksi tutkittiin molekyylipainettujen polymeerien käyttöä mitattavia analyyttejä tunnistavina yhdisteinä. Perinteisiä sensoreissa käytettäviä biomolekyylejä kestävämpinä yhdisteinä ne voisivat soveltua etenkin bioprosessien jatkuvaan seuraamiseen. Tutkimuksessa kehitettiin myös foolihapon tunnistava molekyylipainettu polymeeri, jota käytettiin kiinteän faasin sitoja-aineena mikrofluidisessa detektio- ja uuttojärjestelmässä.

La fonctionnalisation chimique de surfaces hétérogènes (fonctionnalisation orthogonale) est une méthode efficace pour diriger l’ancrage de diverses cibles (biomolécules ou nano-objets) sur des zones précises prédéfinies sur un substrat. Ceci est particulièrement intéressant dans le domaine des biocapteurs à plasmons localisés (LSPR) où la transduction ne peut se faire que sur des nano- structures métalliques. L’enjeu est alors d’assurer que les molécules à détecter se fixent spécifiquement sur ces nanostructures et ne s’adsorbent pas sur la surface diélectrique environnante. Dans ce but, nous avons développé dans cette thèse des fonctionnalisations orthogonales de surfaces micro et nanostructurées d’or sur silice à l’aide de divers thiols et silanes. Par rapport à l’état de l’art dans ce domaine, nous avons notamment proposé un protocole en une seule étape et démontré la bonne orthogonalité de ces fonctionnalisations par différentes méthodes de caractérisation chimique de surface (notamment PM-IRRAS, XPS et ToF-SIMS). De plus, ces fonctionnalisations sélectives ont permis l’ancrage spécifique de diverses nanoparticules de latex sur des micro et nanostructures d’or entourées de silice, démontré par MEB. Actuellement, cette méthodologie est en cours d’application dans deux composants photoniques différents où l’on attend d’une part des effets d’exaltation de fluorescence par couplage de nano-antennes et nanobilles marquées et d’autre part un gain en sensibilité d’un biocapteur LSPR pour la détection de différentes biomolécules. / Orthogonal surface chemical functionalization is an efficient method for the selective trapping of different targets (biomolecules or nano-objects) onto predefined regions of a patterned substrate. This is specially interesting in the field of localized surface plasmon resonance (LSPR) biosensors, where transduction only occurs on metallic nanostructures. The aim is thus to ensure that the target molecules can be selectively anchored onto these nanostructures and not adsorbed on the surroun- ding dielectric surface. Thus, we have developped during this PhD different orthogonal functio- nalizations of micro and nanopatterned gold on silica surfaces with thiols and silanes. In regards to the state of the art in this topic, we have proposed a single-step protocol and demonstrated the good orthogonality of such functionalizations by extensive surface chemical characterization including PM-IRRAS, XPS and ToF-SIMS analysis. Furthermore, these functionalizations have been used for the selective anchoring of different latex nanoparticles onto micro and nanopatterns of gold surrounded by silica, as shown by SEM. At the moment, this methodology is being applied in two different photonic devices where we expect on the one hand a coupling between fluorescent nano- beads and plasmonic nano-antennas and, on the other hand, the increase in sensitivity of an LSPR biosensor for detecting different biomolecules.

M.Sc. (Chemistry) / With the increase in the number of new Human Immunodeficiency Virus (HIV) infection and mortality rate worldwide partly due to diagnostic drawbacks which gives false negatives during the window period, it is imperative to find an alternative method of detection. The need for prompt, bed-side and field applicable analytical devices for biomedical purposes cannot be over emphasized in our fast paced world today. As a contribution to meeting these challenges, this dissertation reports (i) the development of novel electrochemical DNA/RNA aptamer biosensor for HIV sensing and (ii) the development of other DNA sequence specific electrochemical biosensors. These biosensors were based on composite platforms of dendrimer and gold nanoparticles...

With the increased usage and production of engineered nanoparticles (ENPs), entry into the environment and hence contact with plant root systems is inevitable. Nano zinc oxide (nZnO) is widely used in commercial products, such as sunscreens, paints and coatings due to its high antimicrobial properties and wide electrical band-gap. Disposal down drains and into greywater leads to particle entry into the environment via waste water systems. Here, ENPs could potentially interact with plant root systems, which may lead to uptake, translocation and accumulation within plant tissues, and in the case of edible crops have consequences on human health. This study aimed to identify mechanisms of toxicity by employing whole-cell biosensors in conjunction with model bacteria and plant species. Furthermore, zeta potential (ZP), particle size, reactive oxygen species (ROS) release and solubility of the particles were determined and linked to both plant and bacterial toxicity. In Escherichia coli bacteria, it was demonstrated that growth inhibition from nano-scale ZnO treatment was similar to that from the bulk-scale ZnO and ionic zinc treatments, with the concentrations leading to 50 % inhibition (IC50) demonstrated to be 251, 282 and 298 mg/L for bulk, nano-scale and ZnSO4, respectively. It was demonstrated that the mode of nZnO toxicity in E. coli was bacteriostatic rather than bacteriotoxic. In barley plants, biomass was negatively impacted by up to 50 %, and significantly more zinc was able to enter root tissues as a result of hydroponic nZnO treatment, with 47 mg/L zinc detected in root tissues after 7 days treatment with 500 mg/L nZnO. Comparison of particle characteristics revealed that ROS, solubility, ZP, size and concentration were involved in toxicity, with ZP (charge) identified as a key parameter in both plant and bacterial toxicity.

No description available.

L’objection générale de ces travaux est de comprendre les mécanismes d’interaction de molécules d’intérêt avec les membranes afin de faciliter la synthèse de molécules plus efficaces contre leurs cibles tout en étant moins toxique pour l’Homme. Dans la première partie de ces travaux, nous avons étudié les interactions entre ces modèles membranaires et les polyènes antifongiques, connus pour interagir avec les stérols des membranes plasmiques. Nous nous sommes particulièrement intéressés à la Nystatine et à l’Amphotéricine B, deux molécules de structure chimique très proche et actuellement utilisées dans l’industrie pharmaceutique. L’utilisation de différents modèles membranaires et de techniques adaptées a montré que la PhosphatidylEthanolamine avait très vraisemblablement un rôle primordial dans le mécanisme d’interaction de ces molécules avec les membranes. Dans la deuxième partie de ces travaux, nous nous sommes intéressés à l’inhibition de la formation du cristal d’hémozoïne formé lors de la croissance du parasite responsable du paludisme. Ce cristal est formé d’hématine, hautement toxique pour le parasite. L’hématine et l’inhibition de la formation de l’hémozoïne constituent une cible moléculaire idéale pour combattre cette maladie. La chloroquine, la méfoquine et de nouveaux inhibiteurs dérivés de la méfloquine ont été utilisés. L’étude de l’inhibition de la formation du cristal s’est faite en utilisant des monocouches de Langmuir, servant ainsi de biocapteurs. Ces travaux ont montré que l’énantiomérie, mais aussi la lipophilicité des nouveaux composés antipaludiques sont des paramètres importants en vue de la synthèse de molécules plus efficaces. / The main purpose of this work is to better understand the mechanisms of interaction between pharmaceutical relevant molecules and model membranes in order to facilitate the synthesis of new molecules, more efficient against their molecular target and less toxic for Humans. In the first part, we studied the interactions occuring between these models and antifungal polyene molecules. It has been reported that these molecules interacted preferentially with sterols. We specifically focused on Nystatin and Amphotericin B, two polyenes with a very similar chemical structure and presently used as a treatment against fungi and molds. Using different kind of model membranes, we showed PhosphatidylEthanolamine could have a very important role in the mechanism of action of these molecules. In the second part of this work, we studied the inhibition of the formation of a cristal called « hemozoïn », which is growing during the life cycle of the parasite responsible of malaria. This cristal is made of hematin, a toxic by-product of the degradation of hemoglobin, the main source of amino-acids for the parasite. Hematin and the inhibition of the growth of this cristal is a ideal molecular target to combat malaria. Chloroquine, mefloquine and new mefloquine-derivatives were studied. The study of the inhibition of the formation of the cristal was done using Langmuir monolayers as a biosensor. We showed that stereochemistry, but also lipophilicity of these compounds, are important parameters for the synthesis of more efficient antimalarial molecules.

Accurate diagnosis of active tuberculosis is required to improve treatment, reduce transmission of the disease and control the emergence of drug resistance. A rapid and reliable test would make a considerable contribution to the management of the TB epidemic, especially in HIV-burdened and resource-poor countries where access to diagnostic laboratories are limited. Surrogate marker antibody detection to mycobacterial lipid cell wall antigens gave promising results, in particular with cord factor. The specific advantage of using mycolic acids as lipid antigens in comparison to protein antigens is that mycolic acid is a CD1 restricted antigen with the ability to induce proliferation of CD4/CD8 double negative T-cells, which may explain the sustained antibody production in AIDS patients. Traditional end-point assays to detect anti-MA antibodies showed an unacceptable number of false positive and negative test results. Here a much improved biosensor method (the MARTI-assay, i.e. Mycolic acid Antibody Real-Time Inhibition assay) was developed to detect antibodies to mycolic acid in patient sera as surrogate markers of active tuberculosis. The test was assessed on an IAsys optical biosensor and gave an accuracy of 82%. The technology was transferred to an SPR (ESPRIT) biosensor to economise and simplify the assay. Mycolic acid containing liposomes were immobilized on the SPR gold surface pre-coated with octadecanethiol. The following parameters were optimized on the ESPRIT biosensor to enable reliable TB diagnosis: effect of degassed buffer, saponin blocking, first exposure to serum at low concentration and second exposure to antigen inhibited serum at high concentration. The IAsys biosensor system has a weakness in the double channel cuvette system, in which the channels often do not give matching results, while being ten times more expensive than the gold discs provided for the ESPRIT biosensor. The ESPRIT biosensor is provided with an adjustable laser setting to compensate for differences in the channel readings as well as an automated fluidic system that reduces variance from one sample to the next. First indications are that the test can also be used for prognosis of TB during treatment. It is hoped that the ESPRIT biosensor will improve the accuracy of the test to more than 90%. If the MARTI-assay technology could be made amenable for high throughput screening, it may provide the solution to the serodiagnosis of tuberculosis and monitoring of progress during TB treatment both in adult and children, thereby reducing the spread of TB within the communities. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

Developing non-invasive and accurate diagnostics that are easily manufactured, robust and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment, particularly in the developing world. There is currently no rapid, low-cost and generic sensor fabrication technique capable of producing narrow-band, uniform, reversible colorimetric readouts with a high-tuneability range. This thesis aims to present a theoretical and experimental basis for the rapid fabrication, optimisation and testing of holographic sensors for the quantification of pH, organic solvents, metal cations, and glucose in solutions. The sensing mechanism was computationally modelled to optimise its optical characteristics and predict the readouts. A single pulse of a laser (6 ns, 532 nm, 350 mJ) in holographic “Denisyuk” reflection mode allowed rapid production of sensors through silver-halide chemistry, in situ particle size reduction and photopolymerisation. The fabricated sensors consisted of off-axis Bragg diffraction gratings of ordered silver nanoparticles and localised refractive index changes in poly(2-hydroxyethyl methacrylate) and polyacrylamide films. The sensors exhibited reversible Bragg peak shifts, and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 500-1100 nm. The application of the holographic sensors was demonstrated by sensing pH in artificial urine over the physiological range (4.5-9.0), with a sensitivity of 48 nm/pH unit between pH 5.0 and 6.0. For sensing metal cations, a porphyrin derivative was synthesised to act as the crosslinker, the light absorbing material, the component of a diffraction grating, as well as the cation chelating agent. The sensor allowed reversible quantification of Cu2+ and Fe2+ ions (50 mM - 1 M) with a response time within 50 s. Clinical trials of a glucose sensor in the urine samples of diabetic patients demonstrated that the glucose sensor has an improved performance compared to a commercial high-throughput urinalysis device. The experimental sensitivity of the glucose sensor exhibited a limit of detection of 90 µM, and permitted diagnosis of glucosuria up to 350 mM. The sensor response was achieved within 5 min and the sensor could be reused about 400 times without compromising its accuracy. Holographic sensors were also tested in flake form, and integrated with paper-iron oxide composites, dyed filter and chromatography papers, and nitrocellulose-based test strips. Finally, a generic smartphone application was developed and tested to quantify colorimetric tests for both Android and iOS operating systems. The developed sensing platform and the smartphone application have implications for the development of low-cost, reusable and equipment-free point-of-care diagnostic devices.