Novel Microfluidic Tools for Multiplex Colorimetric Detection of Analytes and Point Mutation Detection in Infectious Pathogens

Abstract

Recently, the need for affordable point-of-care tests has grown exponentially. An ideal point of care (POC) test or device should satisfy the WHO-endorsed ASSURED criteria, meaning that the test/device should be accessible, sensitive, specific, user-friendly, rapid, equipment-free, and deliverable to end users. Paper microfluidics has emerged as a powerful tool for POC testing. Paper-based microfluidic devices offer pump-free fluid transport, use less reagent volume, and can store multiple dried reagents for a prolonged period. Paper membranes can be arranged in different combinations to enable single-step or multi-step detection of micro- or macromolecules in any fluid sample. This thesis presents three novel paper-based POC devices for i) multiplex detection, ii) chloride ion quantification, and iii) antimicrobial resistance (AMR) detection biological fluids: saliva, sweat, and sputum, respectively. The assays described in this thesis overcome critical challenges of the existing POC tests in their respective fields. All three devices have simple fabrication methods, require minimal equipment, and enable direct visual readout, and would thus be accessible in limited resource settings. The first paper-based POC device presented enables the multiplex colorimetric detection of dissolved analytes in paper devices, without having to pattern paper with hydrophobic barriers. barrier-free detection of multiplex analytes. The existing microfluidic paper analytical devices (μPADs) enable multiplex detection by patterning the paper with hydrophobic barriers to create flow channels, which requires expensive equipment, posing a challenge for scale-up. The developed paper-based device, called barrier-free μPAD (BF-μPAD), requires no physical or chemical membrane modification for multiplex detection. The device consists of a stack of two paper membranes with different wicking rates; the top layer acts as a fluid-distributing layer, and the bottom layer contains reagents for colorimetric detection. This paper assembly enables faster fluid flow and generates perfectly isolated signal zones, while improving the limit of detection of colorimetric assays by 3.5x compared to conventional μPADs. The multiplexing feature of BF-μPAD is demonstrated for colorimetric detection of thiocyanate, protein, glucose, and nitrite. The device geometry is modeled in COMSOL Multiphysics software using the Richards equation to understand the fluid flow profile that gives rise to uniform signals in the barrier-free assembly. The concept of stacking a fast-wicking paper membrane on top of a slow-wicking paper membrane for uniform rehydration of the dried reagents on the bottom membrane was further utilized for the in-situ synthesis of an insoluble reagent, silver chromate. The bottom paper membrane containing silver chromate was cut into narrow strips to develop a distance-based sensor for sweat chloride quantification. Synthesis of silver chromate was previously accomplished by manually dipping the hydrophobically patterned paper strips into large volumes of precursor solutions with intermittent washing and drying. The present method obviates the need for patterning hydrophobic barriers and eliminates the requirement of multiple dipping steps. The developed sensor has a limit of detection of 0.3 mM and a wide linear dynamic range of 0–120 mM for chloride ion detection, and therefore could be used for the diagnosis of cystic fibrosis, characterized by sweat chloride levels greater than 60 mM. In addition to developing POC devices for sensing chemical and biochemical compounds, we have developed a POC assay for genotypic antimicrobial resistance (AMR) detection, which involves the detection of point mutations in the genome of the organism. An application of the developed bacteria responsible for causing tuberculosis (TB). Drug-resistant TB (DR-TB) is a lethal infection that causes half a million deaths annually. The current genotypic methods for DR-TB detection, e.g., GeneXpert and line probe assays (LPAs), require expensive equipment and skilled personnel, restricting them to a few certified laboratories. Our assay only requires a centrifuge and a thermal cycler, and enables rapid, low-cost, and decentralized DR-TB detection. The assay detects the four most common mutations in the rpoB gene that confer Mtb rifampicin resistant: S531L, H526Y, H526D, and D516V. These mutations are detected using oligonucleotide ligation assay (OLA), and the results are directly visualized on a paper strip, making the assay point of care compatible. The assay reports a clinical sensitivity and specificity of 90.90% and 100%, respectively, for DR-TB detection (N=29) and can detect as low as 3% mutant TB strains in a sample containing a mixture of mutant and wild type strains. The developed method is amenable to be performed at peripheral locations where the infection is most prevalent. We have also developed two alternative strategies to simplify the workflow and further reduce the instrumentation cost of the DR-TB detection assays. The first strategy combines DNA amplification and point mutation detection reaction in a single pot. The combination reduces the assay turnaround time from 3.5 hours to 2 hours. Combining the two reactions required developing a new buffer composition compatible with PCR and OLA. The other strategy is to use loop- mediated isothermal amplification (LAMP) for DNA amplification. Because LAMP amplicons consist of intermittent single-stranded loop regions, the loops act as a template for probes to anneal, enabling isothermal ligation. This strategy would replace the thermal cycler machine with a temperature incubator, further reducing the instrumentation cost for DR-TB detection. A proof of concept of this strategy was demonstrated by performing isothermal ligation on the synthetic loop region of the LAMP assay. Overall, the three main methods described in this thesis are technological advancements in their respective domains that have opened new gates of research in paper microfluidics and POC diagnostics