Date of Award

Summer 8-1-2017

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science in Engineering (MSE)

Department

Biological Engineering

Advisor

Paul Millard

Second Committee Member

Douglas Bousfield

Third Committee Member

Sara Walton

Abstract

The use of the zebrafish as an animal model alternative to mammalian species has spawned research advancements in several medical fields. Since the zebrafish shares a high degree of sequence and functional homology with mammals, studies using this organism can provide in-depth insight into host response to disease and provide a platform for testing a range of treatment options. The optical transparency of zebrafish at early stages of development permits easy assessment of the effects of treatments, occurrence of tumors and other abnormal growth, disease progression, and immune response, to name only a few. These characteristics make it ideal for studying human diseases such as the Influenza A Virus (IAV). Recent research reveals that zebrafish produce sialic acids that are identical to that of humans. IAV possesses receptors that bind directly to sialic acid receptors, permitting infection. The conventional method of IAV transmission is by aerosol, and since the zebrafish does not accommodate this mode of entry, the virus is injected into the specimen under study.

Zebrafish larvae are fragile and susceptible to deformation, therefore handling can be challenging. The larvae typically require manipulation during preparatory procedures prior to assessment, a process that can be time consuming and stressful for the organism.

In this thesis, I describe the development of a device designed to eliminate the problems associated with manipulating zebrafish larvae by automatically conducting specimen from a reservoir directly into an entrapment dock, where it will be immobilized for injection and rapidly removed post-injection. This will help to significantly reduce the handling time of large sample sets, thereby increasing the screening throughput. Zebrafish have fast growth rateshence preparatory procedures for analysis like injection should be as quick and efficient as possible. This will reduce the likelihood that a large group of fish will transition to different stages of development prior to analysis. The device employs a system to conduct 48-72 hour old zebrafish through a liquid medium (egg water) using a syringe pump. The complete system consists of three main subsystems, namely the pump, optical detection and entrapment components. A 3D printed housing encloses the electrical components of the entire system. The device works by aspirating individual fish through a tube via a pressure gradient created with a syringe pump. Each cycle of the device involves the following steps: (1) loading, (2) sensing, (3) trapping, (4) injection, and (5) flushing. During loading, a single larva is extracted from the reservoir and conducted through a tube past the optical detection subsystem. At the sensing stage, the optical detection subsystem composed of a photodiode and a laser, senses transmitted light from the laser and discerns the entry of larva from air bubbles and debris with precision. Upon larva recognition, the specimen is then conducted to the entrapment dock (step 3) where it will be immobilized for injection (step 4). The final step (5) involves conducting the larva out of the entrapment dock and subsequently out of entire system for further analysis. This device will primarily serve IAV researchers who intend to introduce vaccines, pathogens and other experimental materials into many individual zebrafish larvae.

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