| Description |
The effective diagnosis of infectious diseases, like tuberculosis (TB), through the detection of biomarkers indicative of active infection, continues to challenge the scientific community. Due to the consistently high burden of disease in low-income economies, there has been a renewed interest to transition the capabilities of the diagnostic tests from the research laboratory to point-of-need (PON) applications. To identify the characteristics of these PON tests, the World Health Organization has established the ASSURED ( affordable, sensitive, specific, user-friendly, rapid, equipment-free, and delivered to those in need) guidelines. Using the detection of mannose-capped lipoarabinomannan (ManLAM), a TB biomarker, as a model system, the research presented herein focuses on approaches to meet the challenges faced by modern infectious disease diagnostics with an emphasis on transitioning state-of-the-art surface-enhanced Raman scattering (SERS) immunoassays toward PON applications within the framework of the ASSURED guidelines. First, we build on previous work investigating the underpinnings of an acid treatment method to improve the detection of ManLAM in the serum of infected individuals. This work demonstrates that while acid treatment improves the detection of ManLAM, assay performance is hindered because of the acid-induced degradation of ManLAM and the incomplete decomplexation of endogenous serum proteins. Through the application of an enzymatic sample treatment process, this work also shows that improved ManLAM recoveries lead to improved clinical accuracies. To increase assay performance, we developed, charactered, and validated a novel surface-enhanced resonance Raman scattering (SERRS) immunoassay. This improves the limit of detection (∼10x) and analytical sensitivity (∼39x) for ManLAM measurements compared to an analogous SERS immunoassay. The remainder of the work validates the use of a handheld Raman spectrometer for the detection of phospho-myo-inositol-capped LAM (PILAM), a ManLAM simulant. This work demonstrates the ability to achieve low limits of detection (∼0.2 ng/mL) for PILAM in human serum and document the impact of excitation wavelength and the plasmonic coupling between the labels and planar gold substrates as a basis for further improvements in SERS immunoassays. Taken together, this work begins to establish approaches for improved methodologies to combat the burden of infectious diseases, and to demonstrate the applicability of SERS detection beyond the research laboratory. |
