Development of Sensitive Biomolecule Detection Strategies for Low-Resource Settings
Gibson, Lauren Elizabeth
In low-resource areas of the world, accurate diagnosis of disease is especially challenging, as resources are limited and environmental conditions uncontrolled. Consequently, reliable low-resource diagnostics must be simple, cost effective, stable and sensitive. For diagnosis of malaria in low-resource settings, lateral-flow rapid diagnostic tests (RDTs) are commonly used. Unfortunately, RDTs lack sensitivity, leaving many low-level and asymptomatic infections untreated. In this work, new diagnostic strategies that are both stable and sensitive were developed for the malarial biomarker Plasmodium falciparum histidine-rich protein II (pfHRPII). Patient samples were analyzed to gain further insight into the properties of this biomarker. Additionally, sample preparation techniques utilizing metal affinity ligands for capture of this histidine-rich biomarker were evaluated. These methods concentrated pfHRPII from a large sample volumes, allowing for greater biomarker presentation to the tests. Magnetic particles utilizing this this capture strategy were shown to lower detection limits of commercial RDTs to single-digit parasitemias. To eliminate the need for additional diagnostic components, cellulose membranes modified with metal affinity ligands were synthesized. This inexpensive method allowed for capture and detection of pfHRPII on the membrane. Additionally, detection strategies based upon signal amplification with porphyrin nanoparticles were developed. In these methods, the dissolution of each nanoparticle into tens of thousands of porphyrin molecules resulted in amplified detection of the biomarker. By using nanoparticles as the signal-generating moiety, stability of the detection method was increased relative to commonly used enzyme-based assays. A fluorescent assay was designed, where the inherent fluorescent signal of tetra(4-carboxyphenyl) porphyrin could easily be measured after nanoparticle dissolution. Additionally, a catalytic assay, which employed a second type of signal amplification through the catalytic turnover of a substrate by hemin (ferriprotoporphyrin IX chloride), was developed. These methods were shown to detect picomolar levels of malarial biomarkers from complex matrices. By combining the modified cellulose membranes with hemin nanoparticle detection, a flow-through diagnostic was constructed. This diagnostic was shown to detect asymptomatic levels of pfHRPII. Thus, this work produced sensitive and stable diagnostic strategies which show great promise for implementation in low-resource settings.