A Rapid Point-of-Care Test for Malaria Diagnostics in Elimination Settings

Abstract

One of the grand challenges in science and engineering today is the development of innovative technologies to address global health concerns in resource-limited settings. This thesis aims to develop self-driven microfluidicsbased diagnostic tools suitable for detecting malaria at the point-of-care (POC). In particular, the objective is to engineer devices that offer high sensitivity, short turnover time, and are amendable to mass manufacturing. In this thesis, we examine the current diagnostic needs for malaria elimination, and identify gaps and opportunities in existing detection tools for malaria. Next, we provide an overview of new malaria diagnostic technologies that could be suitable for low resource settings. We also examine how innovative technologies such as microfluidics can assist in the global defeat of this ancient scourge. Specific examples are also discussed to illustrate the potential of advancement in technology in compliance to simplicity of use, appropriateness and affordability. We develop microfluidics-based proof-of-concept diagnostic devices that are based on the understanding of an ideal malaria rapid test for the elimination context. Two key challenges are addressed, 1) the improvement of sensitivity, and 2) the integration of an immunodiagnostic assay into a self-driven microfluidics platform. To solve the first challenge, we use immuno-gold silver-staining (IGSS) as a mean to strongly amplify signal intensity. The growth of silver film over time for a model immunoassay is characterized and the optimum development time is in agreement with the literature. Additionally, we also confirm the stability of the assay using IGSS at 24°C and 37 °C. To solve the 2nd challenge, we integrate a number of microfluidic elements on the chip. A highlight of this proof-of-concept is the elegant implementation of fluorescent microbeads that serves a two-fold purpose. First, the beads provide sufficient surface to immobilize capture antibodies (cAbs). Second, as the target analyte is captured, silver is produced and this silver film masks the fluorescence emitted from the core of the beads. The detection method for the device therefore relies on fluorescence attenuation and the analyte concentration being inversely proportional. The integrated, capillary-driven microfluidic chip can accommodate 700 nanoliter of liquids and take 20 minutes to detect 17.1 ng mL-1 rabbit IgG, compared to milliliters of liquids and hours needed to detect 24.6 ng mL-1 rabbit IgG using a standard enzymelinked immunosorbent assay (ELISA) on microtiter plates. The proof-of-concept is further extended to detect the presence of Plasmodium falciparum histidine-rich protein-2 (PfHR2) in human serum. Amongst various types of malaria Plasmodium infections, Plasmodium falciparum (Pf) infection is the most fatal one. Symptoms associated with Pf infections are often misinterpreted with other febrile fevers, which results in available treatments not being administered in time. Therefore, the ability to detect the presence of PfHRP2 at the onset of infection is key. Current malaria rapid tests cannot detect low PfHRP2 concentrations (6 ngmL-1), with the exception of nucleic amplification technologies. We base our work on the same chip design and fabrication procedure as before. Unlike the previous assay developed for rabbit IgG, we further improve the sensitivity of the PfHRP2 detection immunoassay by using secondary antibodies to amplify signal intensity before applying the IGSS technique. Since the core of the microbeads is fluorescent, photobleaching and stability issues often found in fluorescent assays are mitigated. The self-driven microfluidic chip is capable of achieving a limit of detection of less than 6 ng mL-1 PfHRP2 in human serum within 20 minutes. This limit of detection surpasses the requirement needed for the ideal malaria diagnostic tests for the elimination context set out by the Foundation for Innovative New Diagnostics (FIND). Looking towards the future, such chips, especially if manufactured in low cost plastic and used in combination with smartphone-based fluorescence readers, have the potential to drive the widespread adoption of fluorescent bead-based immunoassays using capillary-driven microfluidics for POC diagnostics applications.