Development of fluorescence based probes for disease imaging

A thesis submitted to the Faculty of Science, School of Chemistry at University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy, 2018Microscopy detection has once been the gold standard in the detection of infectious diseases such as tuberculosis and cancer, and therefore improvements in the detection procedure could further enhance sensitivity and restore its use. Fluorescent probes for microscopy detection have been synthesized using organic dyes which have numerous limitations, such as a narrow absorption spectra and a broad emission spectra to name but a few, that impact negatively on assay sensitivity. Use of semiconducting nanomaterials in the design of fluorescent probes for disease detection eliminates the aforementioned limitations and enhances the sensitivity of detection, and also allows for the detection of multiple disease analytes further improving the scope of detection which eliminates misdiagnosis. Moreover, commercially available nanomaterials are synthesized from toxic elements such as cadmium, lead and mercury; using an organometallic synthesis route which further yields nanomaterials not suitable for bio applications. This study is based on the aqueous synthesis of semiconducting nanomaterials for use as fluorophores in the development of fluorescent probes for imaging of cellular materials. Copper and Cobalt sulfide nanomaterials were of interest in our study. They were synthesized via an aqueous synthesis method, using four different capping ligands, namely; glutathione, thioglycolic acid, 18-crown-6 and L-carnosine. Reactions were performed at either 50 and 95 ˚C, using deionized water as a solvent. The optical and structural properties of as-synthesized nanoparticles (NPs) were investigated using UV/Vis absorption spectroscopy (UV-Vis), Photoluminescence spectroscopy (PL), Transmission Electron Microscope (TEM), X-Ray Diffraction (XRD) spectroscopy and Fourier Transform Infrared Spectroscopy (FT-IR). The cytotoxic properties of as-synthesized NPs were investigated using the 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide tetrazolium assay (MTS). Fluorescent probes were constructed by conjugating hypoxia inducible factor-1α (HIF-1α) and the Phosphohistone H2A.X (Phospho H2A.X) monoclonal antibodies to as-synthesized NPs using carbodiimide chemistry. The fluorescent probes were used to target HIF-1α and Phospho H2A.X proteins produced in mammalian cells (Human Embryonic Kidney (Hek293) and HeLa cells) undergoing apoptosis, in vitro. iii The optical properties of as-synthesized NPs depicted absorption spectra that are blue shifted from their bulk counterparts, signifying the formation of small nanoparticles. This is due to quantum size effects. Copper sulfide nanoparticles depicted emission peaks that are red shifted from their respective absorption spectra while cobalt sulfide nanoparticles revealed red shifted emission spectra. TEM images revealed the formation of various morphologies, including spheres, hexagons and rods, in response to the use of different capping ligands, synthesis temperatures and sulfur sources. The size of nanoparticles was greatly influenced by the reaction temperature, with small nanoparticles formed at 50 ˚C while bigger particles were formed at 95 ˚C. The XRD revealed the formation of mixed phases when the temperature of the reaction was set at 50 ˚C. At 95 ˚C, single phases were formed and the reactions were seen to be complete as no evidence of non-reacted material was observed. The interaction of the capping ligands with NPs was confirmed via FT-IR, with various capping ligands employing different functional groups to attach to the surface of NPs, which were not affected by reaction temperature or sulfur source employed. The cytotoxicity of as-synthesized NPs was investigated using an MTS assay. Copper sulfide NPs were seen to be non-toxic at concentrations below 6µg/ml, with a dose dependant decrease in cell viability observed throughout. However, for CuS NPs synthesized at 50 ˚C using SDEDTC, a decline is observed when the concentration of nanoparticles exceeds 3.125 µg/ml. CoxSy NPs were found to be non-toxic to MT-4 cells, with cell viability maintained above 80% even at the highest concentration tested. Fluorescent probes were developed by conjugating antibodies to CuxSy NPs for imaging purposes. A slight shift to higher wavelength was observed in the absorption spectra of CuxSy NPs after the conjugation step, signifying the presence of the antibodies on the surface of nanoparticles. When tested in vitro using cells treated with CoCl2 and doxorubicin, the fluorescent probes were internalised by the cells, but their fluorescence intensity was low when compared to cells treated with nonconjugated nanoparticles. These results demonstrate the feasibility of utilising these nanoparticles in the development of fluorescent probes for biological imaging applications in vitro, with further optimisation.XL201

Biosensors: Design, Development and Applications

The ability to detect even the slightest physiological change in the human body with high sensitivity and accurately monitor processes that impact human nature and their surroundings has led to an immense improvement in the quality of life. Biosensors continue to play a critical role across a myriad of fields including biomedical diagnosis, monitoring of treatment and disease progression, drug discovery, food control and environmental monitoring. These novel analytical tools are small devices that use a biological recognition system to investigate or detect molecules. This chapter covers the design and development of biosensors, beginning with a brief historical overview. The working principle and important characteristics or attributes of biosensors will also be addressed. Furthermore, the basic types of biosensors and the general applications of these biosensors in various fields will be discussed

Evaluating the antimicrobial activity and cytotoxicity of polydopamine capped silver and silver/polydopamine core-shell nanocomposites

Fabrication of bioactive nanomaterials with improved stability and low toxicity towards healthy mammalian cells have recently been a topic of interest. Bioactive metal nanomaterials such as silver nanoparticles (AgNPs) tend to lose their stability with time and become toxic to some extent, limiting their biological applications. AgNPs were separately encapsulated and loaded on the surface of a biocompatible polydopamine (PDA) to produce Ag-PDA and Ag@PDA nanocomposites to unravel the issue of agglomeration. PDA was coated through the self-polymerization of dopamine on the surface of AgNPs to produce Ag-PDA core-shells nanocomposites. For Ag@PDA, PDA spheres were first designed through self-polymerization of dopamine followed by in situ reduction of silver nitrate (AgNO3) without any reductant. AgNPs sizes were controlled by varying the concentration of AgNO3. The TEM micrograms showed monodispersed PDA spheres with an average diameter of 238 nm for Ag-PDA and Ag@PDA nanocomposites. Compared to Ag@PDA, Ag-PDA nanocomposites have shown insignificant toxicity towards human embryonic kidney (HEK-293T) and human dermal fibroblasts (HDF) cells with cell viability of over 95% at concentration of 250 µg/mL. A excellent antimicrobial activity of the nanocomposites was observed; with Ag@PDA possessing bactericidal effect at concentration as low as 12.5 µg/mL. Ag-PDA on the other hand were only found to be bacteriostatic against gram-positive and gram-negative bacteria was also observed.The University of Witwatersrand School of Chemistry, The University of the Witwatersrand Postgraduate Merit Award and the National Research Foundation of South Africa.https://www.journals.elsevier.com/arabian-journal-of-chemistryhj2023Physic

Elucidating the structural properties of gold selenide nanostructures

Please read abstract in the article.Mintek and NRFhttp://rsc.li/njc2020-03-14hj2020Physic

Identification of biomarkers of tumour blood vessels for therapeutic targeting

Cancer is a disease that develops as a result of unregulated cell growth, whereby damaged cells grow and divide uncontrollably forming lumps of tissue called tumours. Tumours can either be benign, wherein the tumour is confined within a single cluster and cannot spread to neighbouring tissues, or malignant wherein the tumour is mixed with normal cells and has the ability to invade and grow in normal tissues at a different site (Plank and Sleeman, 2003). The growth and development of tumours is dependent on a continuous supply of blood to ensure a sufficient stream of nutrients and oxygen to the tumour and also the removal of metabolic waste products (Neal and Berry, 2006; Neufeld and Kessler, 2006; DuBois and Demetri, 2007). This is achieved through a process called angiogenesis, the growth of a new network of blood vessels from pre-existing vessels (Bouis et al., 2006; Bhat and Singh, 2008). The aim of the study was to investigate the expression of biomarkers in vascular tumour endothelial (endothelioma) cells and to correlate the biomarker expression with endothelial cell growth and migration, important processes in angiogenesis and tumour progression. Molecules which interfere with cell function, LY294,002 and PF573,228 were employed in this study. LY294,002 is an inhibitor of Phosphatidylinositol 3-kinase (PI3K) signalling, which is involved in the regulation of cell proliferation, cell growth (size) and survival. PF573,228 is an inhibitor of focal adhesion kinase (FAK). FAK forms a scaffold for the attachment of cells at the extracellular matrix and is an important mediator of cell migration and invasion. The MTT assay was used to determine the viability of the cells after treatment with the drugs. Real time cell analysis was also used to determine the migration and invasion of endothelioma cells in response to basic fibroblast growth factor, a proangiogenic factor. Light and electron microscopy were employed to investigate morphological changes of the cells following treatment, while caspase activity was measured using an enzyme linked immunosorbent assay (ELISA). Array studies were undertaken to investigate the expression of angiogenic markers in control and drug-treated cells. A dose-dependent decrease in cell viability and migration were observed in treated cells, with LY294,002 showing a higher potency compared to PF573,228 in both instances. Further studies were undertaken using LY294,002 due to its potency. Light microscopy studies showed that treated cells displayed signs of apoptosis such as an irregular shape and chromatin condensation attributes which were not observed in non-treated cells. This was confirmed by electron microscopy which further revealed signs of apoptosis such as chromatin condensation and margination as well as membrane blebbing. An increase in caspase activity in treated endothelioma cells also confirmed the apoptotic effects of LY294,002. Alteration of the expression levels of various pro- and anti-angiogenic markers in treated cells was observed. Biomarkers that have been suppressed by LY294,002 in treated cells could offer great insight into cancer progression. Also, these markers can serve as targets for cancer therapy in targeted drug delivery.Kanker is 'n siekte wat as gevolg van ongereguleerde selgroei ontwikkel. Beskadigde selle groei en verdeel sonder beheer en vorm knoppe (tumours) in weefsels. Tumours kan of goedaardig wees, waar die tumour beperk is in 'n enkele eenheid en verspreiding na naburige weefsel nie plaasvind nie, of kwaadaardig waar kanker en normale selle gemeng is, en die moontlikheid bestaan om normale weefsel in te dring en te groei in 'n ander ligging (Plank en Sleeman, 2004). Groei en ontwikkeling van tumours is afhanklik van 'n onafgebroke bloedtoevoer om voldoende nutriente en suurstof te verskaf en om metaboliese afbreek produkte te verwyder (Neal en Berry, 2006; neufeld en Kessler, 2006; duBois en Demetri, 2007). Dié word bereik deur 'n proses genoem angiogenese, gedefinieer as die groei van 'n nuwe netwerk bloedvate van reeds bestaande bloedvate (Bouis et al., 2006; Bhat en Singh, 2008). Die doel van die studie was om die voorkoms van biologiese merkers in vaskulêre tumour endoteelselle (?endothelioma? selle) te ondersoek en dit te korrelleer met endoteel selgroei en migrasie, belangrike prosesse in angiogenese en tumour ontwikkeling. Molekules wat selfunksie hinder, LY294,002 en PF573,228 was in hierdie studie gebruik. LY294,002 inhibeer die fosfatidielinositol 3-kinase (PI3K) seinstelsel, wat betrokke is in die regulering van selproliferasie, selgroei (grote) en oorlewing. PF573,228 is 'n inhibeerder van fokale adhesie kinase (FAK). FAK vorm 'n raam vir die aanhegging van selle by die ekstrasellulêre matriks en is 'n belangrike bemiddellaar van selmigrasie en indringing. Die "MTT" toets was gebruik om die prolifererasie van selle na behandeling met sekere middels te bepaal. "Real time" sel annaliese was ook gebruik om die migrasie en indringing van endoteelselle in response tot basiese fibroblaste groei faktor, 'n proangiogeniese faktor, te bepaal. Lig en elektron mikroskopie was gebruik om die morfologiese sel veranderinge na behandeling te ondersoek. Kaspase aktiwiteit was gemeet deur gebruik te maak van ELISA ("enzyme linked immuno assay") "Array" studies was gebruik om die voorkoms van angiogeniese merkers in kontrole en behandelde selle te bepaal. 'n Dosis afhanklike afname in sel proliferasie en migrasie was opgemerk in behandelde selle, met 'n meer merkbare effek van LY294,002, in dié opsig, in vergelyking met PF573,228 in albei gevalle. Met hierdie abservasie in gedagte was verdere studies beperk tot LY294,002. Ligte mikroskoop studies het bewys dat behandelde endoteelselle tekens van apoptose getoon het bv. oneweredige vorm en kromatien kondensering, eienskappe wat nie in die kontrole selle waargeneem was nie. Dié is bevestig deur middel van elektron mikroskopie waar tekens van apoptose soos kromatien kondensie, marginasie en membraan "blebbing" gesien was. Verdere bevestiging van die apoptotiese vermoë van LY294,002 was die verhoging in kaspase aktiwiteit in behandelde endoteelselle. Veranderings in die voorkomsvlakke van verskeie pro- en anti-angiogeniese merkers vir behandelde selle was waargeneem. Biologiese merkers wat deur LY294,002 onderdruk is in behandelde selle kan groter insig gee in die aard van kanker ontwikkeling. Hierdie merkers kan ook as teikens dien vir kankerterapie in geteikende middellewering.Dissertation (MSc)--University of Pretoria, 2014.tm2016Anatomy and PhysiologyMScUnrestricte

Electrochemical impedimetric immunosensor for the detection of measles-specific IgG antibodies after measles infections

The detection of measles-specific primary antibodies (IgG) using electrochemical impedimetric immunosensors is reported. The optimum conditions for electrode saturation were reached after 40 min for 1 μg ml−1 antibody concentrations. Surface roughness using AFM increased with each immobilization or antigen-antibody reaction step clearly confirming the surface modification and recognition between antigen and antibody. The human serum (HS) and new-born calf serum (NCS) spiked with antigen-specific antibody were studied to mimic the real sample analysis. The HS and NCS sera containing antibodies due to measles exhibited correlation between the increasing antibody serum concentrations and the charge-transfer resistance (electrochemically measured). This work clearly showed the potential use of impedance as the preferred electrochemical method for detecting measles-antibodies in label-free manner.Original publication is available at http://dx.doi.org/10.1016/j.bios.2013.04.02

Application of Raman Spectroscopy in Biomedical Diagnostics

In vivo cellular imaging and in vitro assays or sensors are fundamentally used to study the spatiotemporal interaction of molecules at biological interfaces. The study of these interfaces informs various applications such as diagnostics/detection of foreign materials or processes in the biological system. Raman spectroscopy, an optical, non-destructive, label-free fingerprinting tool offers a wide array of applications in both in vitro and in vivo diagnostics owing to its relatively short acquisition time, non-invasiveness and ability to provide biochemical molecular information. It has been explored in tissue imaging, in vitro diagnosis, DNA/RNA analysis, metabolic accretions, single cell analysis photodynamic therapy, etc. The chapter details the application of the optical Raman platform in the detection and imaging of diseases/tissues. The challenges associated with SERS applications and the future outlook as a biomedical diagnostic tool are also discussed

Electrochemical impedimetric immunosensor for the detection of measles-specific IgG antibodies after measles infections

The detection of measles-specific primary antibodies (IgG) using electrochemical impedimetric immunosensors is reported. The optimum conditions for electrode saturation were reached after 40 min for 1 μg ml−1 antibody concentrations. Surface roughness using AFM increased with each immobilization or antigen-antibody reaction step clearly confirming the surface modification and recognition between antigen and antibody. The human serum (HS) and new-born calf serum (NCS) spiked with antigen-specific antibody were studied to mimic the real sample analysis. The HS and NCS sera containing antibodies due to measles exhibited correlation between the increasing antibody serum concentrations and the charge-transfer resistance (electrochemically measured). This work clearly showed the potential use of impedance as the preferred electrochemical method for detecting measles-antibodies in label-free manner.Original publication is available at http://dx.doi.org/10.1016/j.bios.2013.04.02

Electrochemical impedimetric immunosensor for the detection of measles-specific IgG antibodies after measles infections

The detection of measles-specific primary antibodies (IgG) using electrochemical impedimetric immunosensors is reported. The optimum conditions for electrode saturation were reached after 40 min for 1 μg ml−1 antibody concentrations. Surface roughness using AFM increased with each immobilization or antigen-antibody reaction step clearly confirming the surface modification and recognition between antigen and antibody. The human serum (HS) and new-born calf serum (NCS) spiked with antigen-specific antibody were studied to mimic the real sample analysis. The HS and NCS sera containing antibodies due to measles exhibited correlation between the increasing antibody serum concentrations and the charge-transfer resistance (electrochemically measured). This work clearly showed the potential use of impedance as the preferred electrochemical method for detecting measles-antibodies in label-free manner.Original publication is available at http://dx.doi.org/10.1016/j.bios.2013.04.02

Development of a Versatile Half-Strip Lateral Flow Assay toward the Detection of Rift Valley Fever Virus Antibodies

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease that is caused by the Rift Valley fever virus (RVFV); Bunyaviridae: Phlebovirus. RVF disease can affect several different species, including ruminants, camels and humans and thus present a dual threat to public health and livestock food production in endemic regions. In livestock, the RVFV infection is characterised by an acute hepatitis, abortion and high mortality rates in new-born animals. The current RVF diagnostic techniques have shown good sensitivity. However, they require extensive sample processing and complex instrumentation. Owing to speed, low cost, ease of use, and most importantly, the ability to diagnose diseases at sites where they are managed, lateral flow immunoassays (LFIA) are the most widely used point-of-care (POC) tools for disease diagnosis. In this study, a lateral flow assay (LFA) device that is able to detect antibodies against RVFV, with a minimum detectable concentration of 0.125 mg/mL, was successfully developed. The LFA also successfully detected RVFV antibodies in reference RVFV sera. Protein A (ProA), which has the ability to bind immunoglobulins from different species, was used in the detection probe, giving the developed RVFV LFA potential for multi-species diagnosis