Development of immunosensors for the detection of malaria.

Malaria is a disease of global importance caused by an Apicomplexan Plasmodium parasite and transmitted by adult female Anopheles mosquitoes. Malaria affects approximately 50% of the world’s population causing millions of deaths every year. Mostly affected are pregnant women and children under 5 years of age. Morbidity and mortality rates are on the decline in some areas. Despite control efforts the disease continues to affect productivity. Productivity can be increased by early detection. Methods for malaria detection include blood film microscopy, immunochromatographic, serological and molecular tests. Blood film microscopy shows the highest sensitivity and specificity when used by trained personnel with reliable instruments. It is however time-consuming and cannot be applied as a point-of-care diagnostic method. Two electrochemical immunosensors for malaria biomarkers Plasmodium falciparum histidine rich protein 2 (PfHRP 2) and parasite L-Lactate dehydrogenase (LDH) were developed in this work for the detection and quantification of Plasmodium species. The methods were based on screen-printed gold electrodes (SPGEs) with on board carbon counter and silver /silver chloride (Ag / AgCl) pseudo-reference electrode. The first stage of the work involved comparison by characterization of the bare SPGEs using potassium ferricyanide. Electrochemical techniques were used to compare bare and self-assembled monolayers of mercaptoundecanoic acid (MUA) and 3,3´- dithiodipropionic acid (DTDPA) against bare SPGE. The optimal sensor was then used for antibody attachment. For the second stage of the work, adsorption was investigated for capture antibody immobilization on the SPGE. HuCAL monoclonal antibodies against PfHRP 2 conjugated to the electroactive enzyme horseradish peroxidase (HRP) were then applied for signal generation. Electrochemical measurements were conducted using 3,3´ 5,5´-tetramethylbenzidine dihydrochloride and hydrogen,peroxide (TMB / H₂O₂) as the mediator / substrate system at potential of -0.2 V. The sensors utilized sandwich enzyme-linked immunosorbent assay (ELISA),format with HuCAL monoclonal antibodies against Plasmodium immobilized on the gold working electrode. The developed biosensor was capable of detecting sub-microscopic Plasmodium infection with a linear range from 1 to 100 ng mL⁻¹ and a limit of detection (LOD) as low as 2.14 ng mL⁻¹ and 2.95 ng mL⁻¹ for PfHRP 2 in buffer and serum assays respectively. When compared with AuNP enhanced assays, the LOD was 36 pg mL⁻¹ and 40 pg mL⁻¹.. Another biomarker Plasmodium falciparum parasite Lactate dehydrogenase (LDH) was also investigated and another sensor developed using a sandwich assay similar to the PfHRP 2 sensor, but incorporating different antibodies against LDH. LOD 1.80 ng mL⁻¹ and 0.70 ng mL⁻¹ for LDH was obtained in buffer and serum assays. When compared with AuNP enhanced assays, the LOD was 19 pg mL⁻¹ and 23 pg mL⁻¹ respectively. As part of the work, culture medium supernatant containing PfHRP 2 and LDH was used to compare the immunosensor sensitivity for the pan-malaria antigen LDH. Sensitivity of the immunosensor was compared against commercially available Plasmodium immunochromatographic (ICT) kits: OptiMAL-IT and BinaxNOW Malaria kits. The optimized immuno-electrochemical biosensor detected the antigen at 0.002 % parasitaemia whereas the OptiMAL-IT ICT was only able to detect the LDH antigen when concentrations were of 2% parasitaemia. BinaxNOW ICT detected both the LDH and PfHRP 2 antigens in concentrations of 4% parasitaemia and showed negative reading at 0.5%parasitaemia in both synchronized and asynchronized samples. This study has developed two highly sensitive, portable and low cost malaria immunosensors for the first time on JD SPGEs. LDH immunosensor detects all Plasmodium species while PfHRP 2 immunosensor is specific for the detection of Plasmodium falciparum biomarker. Both immunosensors detect quantifiable, sub-microscopic levels of the biomarkers with sensitivities higher than the ICT tests. The immunosensors are therefore recommended for field trial.PhD in the School of Engineerin

Development of an immunosensor for PfHRP 2 as a biomarker for Malaria detection

Plasmodium falciparum histidine-rich protein 2 (PfHRP 2) was selected in this work as the biomarker for the detection and diagnosis of malaria. An enzyme-linked immunosorbent assay (ELISA) was first developed to evaluate the immunoreagent’s suitability for the sensor’s development. A gold-based sensor with an integrated counter and an Ag/AgCl reference electrode was first selected and characterised and then used to develop the immunosensor for PfHRP 2, which enables a low cost, easy to use, and sensitive biosensor for malaria diagnosis. The sensor was applied to immobilise the anti-PfHRP 2 monoclonal antibody as the capture receptor. A sandwich ELISA assay format was constructed using horseradish peroxidase (HRP) as the enzyme label, and the electrochemical signal was generated using a 3, 3′, 5, 5′tetramethyl-benzidine dihydrochloride (TMB)/H2O2 system. The performance of the assay and the sensor were optimised and characterised, achieving a PfHRP 2 limit of detection (LOD) of 2.14 ng·mL−1 in buffer samples and 2.95 ng∙mL−1 in 100% spiked serum samples. The assay signal was then amplified using gold nanoparticles conjugated detection antibody-enzyme and a detection limit of 36 pg∙mL−1 was achieved in buffer samples and 40 pg∙mL−1 in serum samples. This sensor format is ideal for malaria detection and on-site analysis as a point-of-care device (POC) in resource-limited settings where the implementation of malaria diagnostics is essential in control and elimination efforts

Surface engineered iron oxide nanoparticles generated by inert gas condensation for biomedical applications

Despite the lifesaving medical discoveries of the last century, there is still an urgent need to improve the curative rate and reduce mortality in many fatal diseases such as cancer. One of the main requirements is to find new ways to deliver therapeutics/drugs more efficiently and only to affected tissues/organs. An exciting new technology is nanomaterials which are being widely investigated as potential nanocarriers to achieve localized drug delivery that would improve therapy and reduce adverse drug side effects. Among all the nanocarriers, iron oxide nanoparticles (IONPs) are one of the most promising as, thanks to their paramagnetic/superparamagnetic properties, they can be easily modified with chemical and biological functions and can be visualized inside the body by magnetic resonance imaging (MRI), while delivering the targeted therapy. Therefore, iron oxide nanoparticles were produced here with a novel method and their properties for potential applications in both diagnostics and therapeutics were investigated. The novel method involves production of free standing IONPs by inert gas condensation via the Mantis NanoGen Trio physical vapor deposition system. The IONPs were first sputtered and deposited on plasma cleaned, polyethylene glycol (PEG) coated silicon wafers. Surface modification of the cleaned wafer with PEG enabled deposition of free-standing IONPs, as once produced, the soft-landed IONPs were suspended by dissolution of the PEG layer in water. Transmission electron microscopic (TEM) characterization revealed free standing, iron oxide nanoparticles with size < 20 nm within a polymer matrix. The nanoparticles were analyzed also by Atomic Force Microscope (AFM), Dynamic Light Scattering (DLS) and NanoSight Nanoparticle Tacking Analysis (NTA). Therefore, our work confirms that inert gas condensation by the Mantis NanoGen Trio physical vapor deposition sputtering at room temperature can be successfully used as a scalable, reproducible process to prepare free-standing IONPs. The PEG- IONPs produced in this work do not require further purification and thanks to their tunable narrow size distribution have potential to be a powerful tool for biomedical applications