Microfluidics devices for Drosophila-based drug discovery assays

Abstract

Drosophila melanogaster has long been a preferred model organism for the study of developmental, genetic, and biochemical processes. They have only four chromosomes and a comparably simple morphology. Given that 60% of Drosophila proteins share homologs with humans, this allows for the effective study of biological processes that are of importance in human and vertebrate models. The conventional drug discovery assays using Drosophila are manual and require skill operator, which makes them expensive and unsuitable for high-throughput screening. Hence, technologies to address the existing challenges involved in the conventional Drosophila-based assays (either in embryonic or larva stage) would greatly facilitate drug discovery process. In this thesis, microfabrication and microfluidics engineering approaches have been utilized in Drosophila-based assays due to their potential to obtain high accurate positioning and reagent delivery in a low-cost, rapid and potentially automated manner. At embryonic stage, the first microinjector that allow one to precisely insert a long taper microneedle laterally and at various positions inside the length of the Drosophila embryo (up to 250µm) was developed. Using this device, it was demonstrated that the cardioblast migration velocity is modified in a dose sensitive manner to varying doses of injected Sodium Azide (NaN3). At larva stage, a systematic analysis of various mechanical constrictions incorporated into microfluidic channels were conducted. This could allow one to find an optimized design for rapid mechanical immobilization of larvae for whole- CNS imaging. The optimal immobilization mechanism has been used for immobilization and live-intact fluorescence functional imaging of Drosophila larva's CNS in response to controlled acoustic stimulation using a Genetically Encoded Calcium Indicator (GECI) probe, called GCaMP5. The microfluidics clamps developed could immobilized Drosophila larvae for only whole-CNS imaging and they cannot allow one to capture neuronal responses at single-neuron resolution, which requires stronger immobilization. In this thesis, a simple microfluidic device, which employed an interesting strategy to completely immobilize the brain and the CNS of a live, fully-functioning Drosophila larva was demonstrated. This enables one, for the first time, to imaging throughout these organs at a single neuron resolution. None of the mechanical immobilization methods available currently are capable of immobilization of the brain and the CNS of a live fully functioning Drosophila larva for intact imaging at a single neuron resolution. The application of this platform was not limited to brain and CNS imaging and it can potentially being used to record neuronal events at different organs such as gut, intestine and hearts in a fully intact manner.