UC San Diego

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.Microfluidic paper-based analytical devices (μPADs) are foundational devices for point-of-care testing yet suffer from limitations in regard to their sensitivity and capability in handling complex assays. Here, we demonstrate an airflow-based, evaporative method that is capable of manipulating fluid flows within paper membranes to offer new functionalities for multistep delivery of reagents and improve the sensitivity of μPADs by 100–1000 times. This method applies an air-jet to a pre-wetted membrane, generating an evaporative gradient such that any solutes become enriched underneath the air-jet spot. By controlling the lateral position of this spot, the solutes in the paper strip are enriched and follow the air jet trajectory, driving the reactions and enhancing visualization for colorimetric readout in multistep assays. The technique has been successfully applied to drive the sequential delivery in multistep immunoassays as well as improve sensitivity for colorimetric detection assays for nucleic acids and proteins via loop-mediated isothermal amplification (LAMP) and ELISA. For colorimetric LAMP detection of the COVID-19 genome, enrichment of the solution on paper could enhance the contrast of the dye to more clearly distinguish between the positive and negative results to achieve a sensitivity of 3 copies of SARS-Cov-2 RNAs. For ELISA, enrichment of the oxidized TMB substrate yielded a sensitivity increase of two-to-three orders of magnitude when compared to non-enriched samples – having a limit of detection of around 200 fM for IgG. Therefore, this enrichment method represents a simple process that can be easily integrated into existing detection assays for controlling fluid flows and improving detection of biomarkers on paper. LNPs, with their lipid bilayer structure, protect fragile mRNA, extending circulation and improving cellular uptake. They can be engineered for tissue targeting and controlled release. Mouse models, due to their genetic similarity to humans, are vital for studying LNPs' biodistribution, pharmacokinetics, and therapeutic impact, as well as assessing immune responses. In this thesis, two PDMS microfluidic devices were developed for mRNA encapsulated LNPs, optimizing parameters like size, encapsulation efficiency, and zeta potential. Intraperitoneal and intravenous injections in mouse models quantitatively assessed LNP efficacy and performance. A groundbreaking high-voltage cold plasma device, paired with advanced cooling, revolutionizing biotechnology. This device enables precise detachment of adherent cells, a milestone in tissue engineering and regenerative medicine. Moreover, it enhances protein binding in ELISA assays, promising higher accuracy in diagnostics, biopharmaceuticals, and life sciences research by manipulating protein-surface interactions. This innovation redefines biotech tools, with far-reaching implications.