Piecing Together the Puzzle: Nanopore Technology in Detection and Quantification of Cancer Biomarkers

Cancer is the result of a multistep process, including various genetic and epigenetic alterations, such as structural variants, transcriptional factors, telomere length, DNA methylation, histone–DNA modification, and aberrant expression of miRNAs. These changes cause gene defects in one of two ways: (1) gain in function which shows enhanced expression or activation of oncogenes, or (2) loss of function which shows repression or inactivation of tumor-suppressor genes. However, most conventional methods for screening and diagnosing cancers require highly trained experts, intensive labor, large counter space (footprint) and extensive capital costs. Consequently, current approaches for cancer detection are still considered highly novel and are not yet practically applicable for clinical usage. Nanopore-based technology has grown rapidly in recent years, which have seen the wide application of biosensing research to a number of life sciences. In this review paper, we present a comprehensive outline of various genetic and epigenetic causal factors of cancer at the molecular level, as well as the use of nanopore technology in the detection and study of those specific factors. With the ability to detect both genetic and epigenetic alterations, nanopore technology would offer a cost-efficient, labor-free and highly practical approach to diagnosing pre-cancerous stages and early-staged tumors in both clinical and laboratory settings

Designing Functional Nanocultures for Controlled Microbial Dynamics

While new techniques and platforms have advanced rapidly to enhance our understanding of complex microbial dynamics utilizing culture-independent methods, the same cannot be said for wet-lab culturing techniques. In fact, developments in meta-omics have revealed large bottlenecks in cell-culturing; omics methods can quickly reveal a myriad of information on microbial populations, yet, recapitulating those same microbial populations in a controlled environment is a non-trivial task. Therefore, there is a critical need to understand how the local milieu of microorganisms facilitates their growth in natural environments. Miniaturized cell-culturing techniques and novel microsystems can facilitate the interrogation of microorganisms and their local milieu in well-defined environments. Innovatively designed micro-technologies have shifted the paradigm in conventional microbial culturing. However, many of these technologies do not provide robust platforms for long-term studies, and are further limited by singular application; therefore, do not translate well from lab to clinical applications. Aspiring to design a novel tool for these purposes, our team has developed Nanocultures: nanolitre-sized, double-emulsion, polymeric microcapsules to sequester and cultivate microbial consortia. The nanocultures provide a robust, optically transparent, and high-throughput tool to study cell dynamics. As semi-permeability is a key design parameter, we can manipulate the chemistry of the nanoculture shell to achieve desired functionalities, such as for high-throughput screening and the development of biotherapeutics to reconstitute a disturbed microflora. As such, this thesis aims to explore I) transport and II) mechanical properties of a newly designed polymer, providing a highly functionalized shell with advantageous semi-permeability properties to the encased bacteria, as well as beneficial mechanical properties that allow for targeted lysis of the polymeric shell. Thirdly, this thesis discusses the design of a novel co-culturing platform to enable the growth of mammalian and bacterial cells together and allows for the assessment of cell-cell interactions in a first attempt, and proof-of-concept design of clinically applicable biotherapeutics. Together, these data will pave the way in our understanding of designing optimal nanocultures for applications-based functionality

Design of a Well-Defined Poly(Dimethylsiloxane)-Based Microbial Nanoculture System

Organosilanes contain hydrocarbon-like backbones, allowing them to react with silicone-based agents in the presence of a catalyst and polymerize into membranes with tunable transport and mechanical properties. Owing to their high hydrophobicity, Poly(dimethylsiloxane) (PDMS) membranes, and more particularly, Sylgard® 184, have been used for applications including drug delivery, gas separation, and microfluidics fabrication. However, the undefined composition of the material and its ability to leach out uncured oligomers make its functionalization and usage challenging for many biological applications. This article presents the design of a novel culture system generated using PDMS-based membranes to study microbial dynamics. The microbial culture system that is referred to as “nanoculture” serves to encapsulate and grow microbes in semipermeable membranes. The mechanical properties of the membranes are reinforced through osmotic annealing, which enable the nanocultures to withstand high shear stress similar to environmental conditions while maintaining transport properties essential to microbial communication and growth. The present study lays the foundation for a novel microbial culture system that would enable the cultivation of microorganisms in environments other than laboratory conditions.</p