PhD Thesis Defence: Ayushi Chauhan

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

Gradual augmentation in lifestyle has directed people to smart technology. The demand for rapid and affordable sample-in answer-out devices for point-of-care (POC) applications has grown exponentially in the past two decades. The inspiration to develop fully integrated POC devices paved the way for paper microfluidics. Paper is an ideal substrate for POC testing owing to its portability, biocompatibility, user-friendliness, and ability to provide pump-free fluid transport.  We present three novel paper-based POC devices for multiplex detection, chloride ion quantification, and antimicrobial resistance (AMR) detection in biological fluids: saliva, sweat, and sputum, respectively. The assays described overcome critical challenges of the existing POC tests in their respective fields. All three devices have simple fabrication methods, require minimal equipment, enable rapid and direct visual readout, and are thus accessible in limited resource settings. The first device described enables multiplex colorimetric detection of analytes in fluids. Existing microfluidic paper analytical devices (μPADs) for this purpose require patterning hydrophobic barriers to create isolated reaction zones, which uses cumbersome techniques. The developed device called barrier-free μPAD (BF-μPAD) obviates the need for patterning barriers. This is accomplished by stacking a fast-wicking paper layer on top of a slow-wicking paper layer, the latter containing dried reagents for analysis. When fluid is introduced into the stack from the top layer, it rapidly distributes through the top layer and generates geographically isolated colorimetric signals in the bottom layer, despite the lack of physical barriers. BF-μPADs improve the limit of detection of colorimetric assays by ~3.5x compared to conventional μPADs. The multiplexing feature of BF-μPADs is demonstrated for colorimetric detection of salivary thiocyanate, protein, glucose, and nitrite.

The concept of stacking paper layers of two different wicking rates was further utilized for the spatially uniform in-situ synthesis of an insoluble reagent, silver chromate, over a large surface area. The in-situ synthesis of silver chromate was previously accomplished by manually dipping the hydrophobically patterned paper strips sequentially 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 paper layer containing silver chromate was cut into narrow strips to develop a distance-based sensor for sweat chloride quantification for cystic fibrosis diagnosis. In addition to developing POC devices for sensing chemical and biochemical compounds, we developed a POC assay for genotypic antimicrobial resistance (AMR), which entails the detection of specific point mutations associated with resistance to known antibiotics. An application of the developed assay is demonstrated for detecting point mutations in Mycobacterium Tuberculosis (Mtb), the bacteria responsible for causing tuberculosis (TB). The current genotypic methods for drug-resistant TB (DR-TB) detection like GeneXpert and line probe assays (LPAs) require expensive equipment and skilled personnel, restricting them to a few certified laboratories. Compared to GeneXpert, our assay significantly reduces the instrumentation cost for point mutation detection by which may enable rapid and decentralized DR-TB detection. The assay detects the four most common mutations in the rpoB gene that confer Mtb rifampicin resistance: S531L, H526Y, H526D, and D516V. The mutations are detected using a molecular technique known as oligonucleotide ligation assay (OLA), and the results are directly visualized on a paper strip, making the assay POC-compatible. The assay reports a clinical sensitivity and specificity of 90.90% and 100% for DR-TB detection (N=29) and can detect as low as 3% mutant TB strain in a pool having 97% regular TB infection.

Two additional exploratory strategies to simplify the workflow and further reduce the instrumentation cost of the DR-TB detection assay are presented. The first strategy combines PCR and OLA reactions in a single pot. The combination reduces the total assay time from 5 hours to 1.5 hours. The other strategy is to use the loop-mediated isothermal amplification (LAMP) method for DNA amplification. Because LAMP amplicons consist of intermittent single-stranded loop regions, the loops act as a template for ligation of, obviating the need for heat denaturation of the target. A proof of concept for isothermal ligation on a synthetic loop region comprising the mutations conferring rifampicin resistance has been demonstrated. This strategy would replace the thermal cycler machine with a temperature incubator, reducing the instrumentation cost for DR-TB detection. Overall, the three main methods described in the thesis are technological advancements in their respective domains that not only open new directions for further research but present opportunities to build commercial products for POC diagnostics.