A Simple Micromilled Microfluidic Impedance Cytometer with Vertical Parallel Electrodes for Cell Viability Analysis

Microfluidic impedance cytometry has been demonstrated as an effective platform for single cell analysis, taking advantage of microfabricated features and dielectric cell sensing methods. In this study, we present a simple microfluidic device to improve the sensitivity, accuracy, and throughput of single suspension cell viability analysis using vertical sidewall electrodes fabricated by a widely accessible negative manufacturing method. A microchannel milled through a 75 µm platinum wire, which was embedded into poly-methyl-methacrylate (PMMA), created a pair of parallel vertical sidewall platinum electrodes. Jurkat cells were interrogated in a custom low-conductivity buffer (1.2 ± 0.04 mS/cm) to reduce current leakage and increase device sensitivity. Confirmed by live/dead staining and electron microscopy, a single optimum excitation frequency of 2 MHz was identified at which live and dead cells were discriminated based on the disruption in the cell membrane associated with cell death. At this frequency, live cells were found to exhibit changes in the impedance phase with no appreciable change in magnitude, while dead cells displayed the opposite behavior. Correlated with video microscopy, a computational algorithm was created that could identify cell detection events and determine cell viability status by application of a mathematical correlation method

Daily magnesium fluxes regulate cellular timekeeping and energy balance

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes1, 2, 3. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology4, 5. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg2+]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago6. Given the essential role of Mg2+ as a cofactor for ATP, a functional consequence of [Mg2+]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg2+ availability has potential to impact upon many of the cell’s more than 600 MgATP-dependent enzymes7 and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR8 is regulated through [Mg2+]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease

Evaluation of Cell Concentration and Viability By Impedance Spectroscopy On Microfluidic Devices

This document describes two distinct platforms that implement electrochemical impedance spectroscopy (EIS) within microfluidic devices for rapid, label-free cell analysis. Each study provides proof-of-concept evaluations of these devices for cell counting and viability analysis applications to mitigate some of the challenges associated with conventional methods. Chapter one includes background information on each version of EIS selected and motivations for the studies conducted. Chapter two describes the design and fabrication of a modular, reusable microfluidic device. Additionally, the methodology for and results from the application of this platform for the measurement of zebrafish sperm cell concentrations are presented. Chapter three describes a microfluidic impedance flow cytometer created by a computer-aided manufacturing method for parallel electrode geometry fabrication. This device was used for single-cell viability testing of Jurkat cells on a continuous flow basis. Cell detection events and discrimination of intact and disrupted cells on the basis of their membrane properties was performed using a custom Matlab script. Major contributions to this project were made by Dr. Julianne Audiffred and Micah Fincher including device design and fabrication, maintenance of cell lines, and raw signal collection, that are shown in Dr. Audiffred\u27s dissertation “Quantitative Macro- and Microscale Methods for Characterizing Cell Viability” (\cite{audiffredQuantitativeMacroMicroscale}). My contribution to this project, as detailed in Chapter 3, was to perform a morethorough COMSOL simulation of parallel versus coplanar electrode geometry performance in impedance cytometry applications, and to create a signal processing algorithm to re-analyze the raw experimental data to improve upon the work pursuant to viability status discrimination. As such, this work will be the basis of a co-authored manuscript that has been significantly re-written to include comparisons with microfluidic impedance cytometry devices that have published more recently. Chapter four includes a summary of conclusions from these efforts and a discussion of proposed future directions

Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species

Accurate determination of sperm concentration in aquatic species is important for assisted reproduction and cryopreservation, yet is challenging as current counting methods are costly or not suitable for many species. The goal of this work was to develop a simple (single-piece and single-layer photolithography) sperm counting chamber (SSCC) for aquatic species. Goldfish ( and zebrafish () sperm were used for evaluation in the device, which was created with soft lithography. Four designs with different geometries were evaluated for counting accuracy. Open-corner and open-midpoint designs were the most accurate with no significant differences ( \u3e 0.05) for most of the target sperm concentrations (0.5-1.0 × 10 cells/mL). The open-corner design was not significantly different from the Makler counting chamber intended for human sperm cells ( = 0.6) but was significantly different from a hemocytometer ( \u3c 0.001) intended for other cell sizes. Material cost of device production was USD 16 per unit, including photolithography supplies, glass slide and coverslip, and polydimethylsiloxane. The cost can be reduced to USD 2 per unit with repeated wafer casts. This device could be further refined for resin 3-D printing and sharing via open-hardware approaches and modified to best suit species specific applications

Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species

Accurate determination of sperm concentration in aquatic species is important for assisted reproduction and cryopreservation, yet is challenging as current counting methods are costly or not suitable for many species. The goal of this work was to develop a simple (single-piece and single-layer photolithography) sperm counting chamber (SSCC) for aquatic species. Goldfish (Carassius auratus) and zebrafish (Danio rerio) sperm were used for evaluation in the device, which was created with soft lithography. Four designs with different geometries were evaluated for counting accuracy. Open-corner and open-midpoint designs were the most accurate with no significant differences (P > 0.05) for most of the target sperm concentrations (0.5–1.0 × 108 cells/mL). The open-corner design was not significantly different from the Makler® counting chamber intended for human sperm cells (P = 0.6) but was significantly different from a hemocytometer (P < 0.001) intended for other cell sizes. Material cost of device production was USD 16 per unit, including photolithography supplies, glass slide and coverslip, and polydimethylsiloxane. The cost can be reduced to USD 2 per unit with repeated wafer casts. This device could be further refined for resin 3-D printing and sharing via open-hardware approaches and modified to best suit species specific applications

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 μm×100 μm) and a PDMS base platform to create a known volume with a 10-μm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations

Genetic Analysis of Impaired Trimethylamine Metabolism Using Whole Exome Sequencing -- Manuscript Draft

Exome sequencing of TMAU patients reveal genetic variation beyond the FMO3 gen

Microbially-Enhanced Vanadium Mining and Bioremediation Under Micro- and Mars Gravity on the International Space Station

As humans explore and settle in space, they will need to mine elements to support industries such as manufacturing and construction. In preparation for the establishment of permanent human settlements across the Solar System, we conducted the ESA BioRock experiment on board the International Space Station to investigate whether biological mining could be accomplished under extraterrestrial gravity conditions. We tested the hypothesis that the gravity (g) level influenced the efficacy with which biomining could be achieved from basalt, an abundant material on the Moon and Mars, by quantifying bioleaching by three different microorganisms under microgravity, simulated Mars and Earth gravitational conditions. One element of interest in mining is vanadium (V), which is added to steel to fabricate high strength, corrosion-resistant structural materials for buildings, transportation, tools and other applications. The results showed that Sphingomonas desiccabilis and Bacillus subtilis enhanced the leaching of vanadium under the three gravity conditions compared to sterile controls by 184.92 to 283.22%, respectively. Gravity did not have a significant effect on mean leaching, thus showing the potential for biomining on Solar System objects with diverse gravitational conditions. Our results demonstrate the potential to use microorganisms to conduct elemental mining and other bioindustrial processes in space locations with non-1 × g gravity. These same principles apply to extraterrestrial bioremediation and elemental recycling beyond Earth