Early Detection of Malaria Parasitic RNA Through an Amplification-Free Surface-Enhanced Raman Spectroscopy Biosensor
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Abstract
Malaria retains its position as one of the most highly transferable diseases throughout the world today, with current efforts focused on controlling its spread. A sensitive, asymptomatic-stage detection method is needed to diagnose malaria before patients begin to experience symptoms. This asymptomatic-stage method would detect the malaria parasite within its early gametocyte stage, before transmission can occur. This would eliminate transference between hosts and greatly reduce malaria cases around the world. However, the current primary diagnostic techniques for malaria detection centers around the symptomatic stage. Light microscopy is the gold standard for malaria diagnostics as it requires low cost and a quick turnaround time; however, this technique lacks sensitivity and requires skilled personnel to visualize the malaria parasite within the blood sample. Rapid diagnostic tests (RDTs), which detect malaria antigens within fifteen minutes, are also available. While these are time efficient and do not require skill to perform, they can only detect symptomatic stage malaria, similar to light microscopy, as RDTs require a high number of malaria parasites in the blood sample to be detectable. Lastly, polymerase chain reaction (PCR) can be used to detect parasitic malaria RNA sensitively but requires an extensive laboratory setup which proves to be expensive and unreasonable for in-field applications. Surface Enhanced Raman Spectroscopy (SERS) has emerged as a useful analytical method that possesses high sensitivity and selectivity. With the exploitation of SERS, this assay aims to produce a technique for asymptomatic-stage malaria detection which will be portable for point-of-care (POC) application. This system targets the Pfs25-mRNA malaria biomarker, which is linked to gametocyte detection. We use a sandwich hybridization framework to employ reporter and capture probes with complementary nucleic acid sequences to bind to the target RNA sequence. The reporter probe is composed of a silver-coated gold nanostar (Ag@AuNS) or a silicon-coated gold nanostar (Si@AuNS) with an embedded Raman tag to serve as a signal reporter while providing SERS enhancement from the plasmonic, noble metal properties of the silver and gold. This thesis compares the enhancement achieved from utilization of rose bengal (RB), cyanine5 (Cy5), and cyanine7 (Cy7) dyes as the embedded Raman tag. A gold-coated silver nanostar (Au@AgNS) complex is also investigated as an optimized SERS substrate for this and other SERS-based assays. The capture probe currently involves the use of a magnetic bead, which is used to pull the hybridization of the three sequences out of the biological matrix. This thesis also investigates the use of a capture probe stick in comparison to the magnetic bead capture probe for a more user-friendly kit. Lastly, we aim to streamline this assay into an integrated kit consisting of a portable Raman spectrometer and the development of a machine learning model to deliver diagnostic result status through a thresholding software. Overall, this assay utilizes the sensitivity and specificity of SERS along with the portability it provides to develop a novel, practical malaria diagnostic technique which will reduce the transmission of the disease worldwide and be of use in POC application.