Droplet microfluidics for digital quantification and multistep assays

Abstract

The ability to detect and quantify viral nucleic acids is of paramount significance for diagnostics, therapeutics, and vaccine development. To combat globally spreading infectious diseases such as SARS-CoV-2, Ebola, and Zika, rapid and sensitive nucleic acid detection and quantification methods are in grave need for development. Compared with conventional tube-based assays, the digital format of bulk assays (e.g., digital PCR or digital LAMP) holds the potential of enabling absolute quantification of nucleic acids with no dependency on endogenous references or standards. Unlike hot-start digital PCR and LAMP, the implementation of digital quantification of the near-ambient-temperature assays using RPA and CRISPR may suffer from premature target amplification. Droplet microfluidics offers versatile yet controllable handling of samples and reagents, which provides confined compartments for enhanced assays kinetics and can be exploited to effectively circumvent premature amplification before droplet partition. In this thesis, we aim to develop effective yet facile microfluidic technology for reagent handling and manipulation with droplets. Firstly, we developed a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) assay for ultrasensitive nucleic acid quantification. PADLOCK is realized through partitioning MgOAc-deprived RPA assays into picolitre water-in-oil droplets, the digital reaction unlocking is assisted by a downstream microfluidic injector loaded with the reaction initiator MgOAc. Hence, premature amplification in bulk is handily circumvented. To provide an in-depth view, we investigated three sensing schemes including CRISPR-Cas13a sensing, exo probe sensing, and EvaGreen sensing with PADLOCK-CRISPR and PADLOCK-EXO achieving sensitivity at single molecule level. We further demonstrated the utility of PADLOCK-CRISPR assay for HPV16 viral loads quantification in clinical samples, which generated 100% concordant results with qPCR. We believe the thorough investigation provides the community with in-depth insights regarding digital RPA based assays. The novel digital reaction unlocking approach sheds light on the enormous potentials of advancing diagnostics by exploiting droplet microfluidics. Secondly, we modified the previous microfluidic injector, and developed a multiplexable microfluidic injector platform which exploits a novel concentric design for on-demand droplets injection, barcoding and tracking. In our device, the channel is curved along circular electrode to obtain a uniform electric field for injection expansion. By manipulating the operating pressure, droplet sequences can be deterministically injected following any self-defined patterns. We presented various modes of droplets coding and decoding for droplet tracking by incorporating fluorescence as barcode. Moreover, we presented a device with three individually controllable injectors, various single and combinatorial injection modes were demonstrated using designated color codes. The platform is compact, versatile, and easy to integrate. We foresee it can potentially facilitate applications such as multiplexed compound screening, combinatorial synthesis, single cell studies or other multistep assays. Besides, we analyzed droplet sorting process based on the dielectrophoresis (DEP) phenomenon and investigated various parameters’ influences on droplet sorting outcome. Further, we developed a droplet sorting system consisting of an optical module, an electrical module, and a microfluidic sorting chip. We demonstrated various sorting regimes corresponding to different operating conditions. Using our platform, droplet can be successfully sorted in a high-throughput manner based on fluorescence signals. Finally, we developed an instrument-free high-throughput droplet formation device for portable, all-in-one nucleic acid quantification. This thesis presents droplet microfluidic technology to address the challenges in enabling isothermal approaches for highly sensitive, specific, and precise nucleic acids quantification. The developed methods not only offer novel functionalities for the realization of new assay workflow but also promise the feasibility of system integration and automation towards the next-generation diagnostics.

Date of Award	2021
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Shuhuai YAO (Supervisor)