PhD Thesis Defence: Priyanka Agarwal

Advancements in Nucleic Acid Lateral Flow Assays (NALFA).

In recent decades, the demand for rapid and precise nucleic acid amplification tests (NAATs) has grown significantly, driven by the need to address pandemics like Covid-19 and diagnose infectious diseases such as tuberculosis and malaria. Paper-based devices offer a practical solution for disease diagnosis, particularly in regions with limited resources or where advanced laboratories are scarce. Lateral flow assays (LFAs), resembling pregnancy test strips, emerge as a feasible detection method due to their rapidity and user-friendliness. While most LFAs are designed to conduct immunoassays, they have been adapted to detect nucleic acids as well; such LFAs are referred to as nucleic acid lateral flow assays (NALFAs). NALFAs have proven to be a robust tool for detecting amplified NAAT products using minimal instrumentation. Nonetheless, despite their utility, NALFAs have not gained the same popularity as lateral flow immunoassays, and consequently, their commercial adoption has been limited. This work aims to overcome this gap. To improve mechanistic understanding of NALFA, we developed a mathematical model of NALFA that incorporates its key transport phenomena and chemical reactions. Subsequently, we introduce two key advancements: firstly, a novel strategy that provides very high sensitivity and specificity for nucleic acid detection, and secondly, Hot-NALFA, a method that enables the detection of point mutations. To date, the design of NALFAs has primarily employed a black box approach; most researchers have adopted a few published protocols without knowledge of the factors that affect its performance. In this work, we recognize multiple factors that affect the performance of NALFAs and provide a mechanistic explanation for them by utilizing a mathematical model. An important outcome of this work is the understanding that unreacted PCR primers inhibit the signal in NALFA, which necessitates that PCR be run till the end point before utilizing NALFA as a readout method. We also highlight the hook effect that reduces the NALFA signal and prove that this effect necessitates the dilution of amplicons prior to NALFA, as is commonly reported in NALFA protocols. This result has important implications in designing integrated devices that aim to directly couple a PCR reaction to a NALFA, where dilution of amplicons may not be feasible.

Two approaches were developed to obviate the requirement for dilution of amplified products before their introduction onto NALFA. This advancement facilitates the direct connection of an amplification reaction with a NALFA. The first approach involves the modification of the sample pad, where the sample is first added on NALFA to capture the excess of amplicons and unreacted primers. The second approach is centered on diminishing the production of bi-labeled products from PCR, which was achieved by introducing only a fraction of labeled primers, in contrast to the conventional practice of using all labeled primers. In nucleic acid detection through NALFA, prior amplification of target DNA is necessary, commonly achieved using the polymerase chain reaction (PCR) method. However, coupling PCR products with the prevalent ‘Universal NALFA’ designed for the detection of biotin and FITC bi-labelled molecules is problematic due to the inevitable formation of bi-labeled primer dimers. These bi-labeled primer dimers lead to false positive signals, compromising the reliability of PCR-NALFA. We introduce a novel approach integrating Linear-After-The-Exponential PCR (LATE-PCR) with Universal NALFA. LATE-PCR, an advanced form of asymmetric PCR, yields high amounts of single-stranded DNA (ssDNA). The process involves generating biotin-labeled ssDNA through LATE-PCR and hybridizing it with a complementary FITC-labelled probe. The resultant bi-labeled product can be accurately detected on a universal NALFA. This novel method effectively mitigates false signals stemming from bi-labeled primer dimers. Unlike traditional approaches, the primer dimers formed in this context are not bi-labeled, consequently evading detection on the assay. We compared our method with a CRISPR-based NALFA format. Our strategy has 100 times higher analytical sensitivity than the CRISPR-based method.

Point mutations refer to single nucleotide changes in nucleic acid sequences and their detection is crucial for genotypic antimicrobial resistance (AMR) and accurate disease diagnosis. Molecular beacons are widely embraced tools for point mutation detection in PCR-based methods, uniquely capable of differentiating between wild-type and mutant DNA within a specific temperature range. We substituted molecular beacons for linear probes to differentiate wild and mutant DNA on NALFA. However, the molecular beacon exhibited binding affinity to both wild-type and mutant targets at room temperature. Consequently, the test line appeared for both DNA sequences, impairing the accuracy of point mutation detection. We elevated NALFA’s temperature using a custom heating device to overcome this, ensuring precise point mutation detection with molecular beacon specificity. We demonstrated that point mutation can be detected on a universal NALFA without requiring additional enzymes/proteins and with fewer steps than the other existing methods.  An additional study involved the manipulation of fluid velocity across the nitrocellulose membrane within a NALFA, achieved by altering the geometry of the wicking pad. Generally, it was observed that wider wicking pads (in the case of rectangular shapes) or divergent geometries exhibited higher fluid velocities compared to the conventional size.  This thesis showcases technological advancements in NALFAs, enhancing their capabilities, providing deeper insights into their mechanism, and introducing innovative approaches for integrating amplified products and detecting point mutations