Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

Lateral flow tests (LFTs) had a pivotal role in combating the spread of the SARS-CoV-2 virus throughout the COVID-19 pandemic thanks to their affordability and ease of use. Most of LFT devices were based on nitrocellulose membrane strips whose industrial upscaling to billions of devices has already been extensively demonstrated. Nevertheless, the assay option in an LFT format is largely restricted to qualitative detection of the target antigens. In this research, we surveyed the potential of UV nanoimprint lithography (UV-NIL) and extrusion coating (EC) for the high-throughput production of disposable capillary-driven, foil-based tests that allow multistep assays to be implemented for quantitative readout to address the inherent lack of on-demand fluid control and sensitivity of paper-based devices. Both manufacturing technologies operate on the principle of imprinting that enables high-volume, continuous structuring of microfluidic patterns in a roll-to-roll (R2R) production scheme. To demonstrate the feasibility of R2R-fabricated foil chips in a point-of-care biosensing application, we adapted a commercial chemiluminescence multiplex test for COVID-19 antibody detection originally developed for a capillary-driven microfluidic chip manufactured with injection molding (IM). In an effort to build a complete ecosystem for the R2R manufacturing of foil chips, we also recruited additional processes to streamline chip production: R2R biofunctionalization and R2R lamination. Compared to conventional fabrication techniques for microfluidic devices, the R2R techniques highlighted in this work offer unparalleled advantages concerning improved scalability, dexterity of seamless handling, and significant cost reduction. Our preliminary evaluation indicated that the foil chips exhibited comparable performance characteristics to the original IM-fabricated devices. This early success in assay translation highlights the promise of implementing biochemical assays on R2R-manufactured foil chips. Most importantly, it underscores the potential utilization of UV-NIL and EC as an alternative to conventional technologies for the future development in vitro diagnostics (IVD) in response to emerging point-of-care testing demands.