Files
Abstract
Malaria is an infectious disease that affects many lives around the world and eradication efforts are focused on controlling the disease transmission; which requires early diagnosis of the infection before the onset of symptoms, i.e. at the asymptomatic stage. Blood smear microscopy is used for symptomatic malaria diagnosis, but it lacks the specificity and sensitivity required for asymptomatic diagnosis. Alternatively, polymerase chain reaction (PCR) can be used to detect the malaria (plasmodium) parasitic nucleic acids with high sensitivity and specificity, but the point-of-care (POC) implementation of PCR is not practical due to laborious procedures and expensive instrumentation. Recently, Surface Enhanced Raman Spectroscopy (SERS) has become a prominent analytical technique, because of its high molecular specificity and enhanced analytical sensitivity. The overall goal of this thesis is to develop a novel PCR amplification-free, SERS-based approach that can detect an asymptomatic malaria biomarker (Pfs25-mRNA) with sufficient sensitivity for POC applications. We developed a sandwich hybridization assay that captures Pfs25-mRNA target sequence using two partially complementary synthetic nucleic acid sequences attached to nanoparticles known as probes. One probe consists of a plasmonic core-shell nanoparticle with an embedded Raman tag to act as a SERS signal reporter. The other probe involves a magnetic bead allowing the separation of the captured Pfs25-mRNA from the blood matrix. Our work demonstrates the ability of the developed SERS hybridization assay to specifically detect synthetic Pfs25-mRNA in buffer and spiked-in blood lysate without the need for amplification techniques. Along with development of the hybridization assay, a novel gold coated-silver nanostar substrate was developed for potential use in SERS-based applications.