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University of Technology Sydney

13 ARTICLES PUBLISHED IN JoVE

Chemistry

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Sercan Pravadali-Cekic¹, Danijela Kocic¹, Stanley Hua¹, Andrew Jones¹, Gary R. Dennis¹, R. Andrew Shalliker¹

¹School of Science and Health, University of Western Sydney

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

JoVE Core

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Andrew Jones¹, Sercan Pravadali-Cekic¹, Stanley Hua¹, Danijela Kocic¹, Michelle Camenzuli², Gary Dennis¹, Andrew Shalliker¹

¹School of Science and Health, University of Western Sydney, ²Van′t Hoff Institute for Molecular Sciences, University of Amsterdam

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

Chemistry

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Sercan Pravadali-Cekic¹, Danijela Kocic¹, Stanley Hua¹, Andrew Jones¹, Gary Dennis¹, Andrew Shalliker¹

¹School of Science and Health, University of Western Sydney

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

Medicine

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Dominic J. Hare^1,2, Kai Kysenius³, Bence Paul⁴, Beate Knauer^5,6, Robert W. Hutchinson⁷, Ciaran O'Connor⁷, Fred Fryer⁸, Tom P. Hennessey⁸, Ashley I. Bush², Peter J. Crouch³, Philip A. Doble¹

¹Elemental Bio-imaging Facility, University of Technology Sydney, ²Florey Institute of Neuroscience and Mental Health, The University of Melbourne, ³Department of Pathology, The University of Melbourne, ⁴School of Earth Sciences, The University of Melbourne, ⁵Research School, Ruhr University, ⁶Department of Physiology, Monash University, ⁷ESI Ltd., Bozeman, ⁸Agilent Technologies, Mulgrave

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Biochemistry

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Matthew B. O'Rourke^1,2, Matthew P. Padula², Caine Smith³, Priscilla Youssef⁴, Stuart Cordwell¹, Paul Witting⁴, Greg Sutherland³, Ben Crossett¹

¹Mass Spectrometry Core Facility, University of Sydney, ²Proteomics Core Facility, University of Technology Sydney, ³Neuropathology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney, ⁴Redox Biology Group, Discipline of Pathology, School of Medical Sciences, University of Sydney

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

Immunology and Infection

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

Alba Llibre^*1,2, Vincent Bondet^*1,2, Mathieu P. Rodero³, David Hunt⁴, Yanick J. Crow^3,5, Darragh Duffy^1,2

¹Immunobiology of Dendritic Cells, Institut Pasteur, ²INSERM U1223, ³Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, ⁴MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, ⁵Manchester Centre for Genomic Medicine, University of Manchester

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples.

JoVE Core

Quantifying the Relative Thickness of Conductive Ferromagnetic Materials Using Detector Coil-Based Pulsed Eddy Current Sensors

Nalika Ulapane¹, Karthick Thiyagarajan², David Hunt², Jaime Valls Miro²

¹Melbourne School of Engineering, University of Melbourne, ²Center for Autonomous Systems, University of Technology Sydney

Here, we present a protocol to quantify the relative thickness (i.e., thickness as a percentage with respect to a reference) of conductive ferromagnetic materials using detector coil-based pulsed eddy current sensors, while overcoming the calibration requirement.

Chemistry

Luminescence Lifetime Imaging of O₂ with a Frequency-Domain-Based Camera System

Maria Moßhammer^*1, Vincent V. Scholz^*2, Gerhard Holst³, Michael Kühl^1,4, Klaus Koren⁵

¹Marine Biological Section, Department of Biology, University of Copenhagen, ²Center for Electromicrobiology, Aarhus University, ³PCO AG, ⁴Climate Change Cluster, University of Technology Sydney, ⁵Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience, Aarhus University

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O₂ distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O₂ microenvironment in the rhizosphere of aquatic plants.

Environment

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Estelle E. Clerc¹, Jean-Baptiste Raina², Bennett S. Lambert³, Justin Seymour², Roman Stocker¹

¹Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, ETH Zürich, ²Climate Change Cluster, University of Technology Sydney, ³School of Oceanography, University of Washington

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Biology

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Amani Alghalayini^1,2, Lele Jiang¹, Xi Gu³, Guan Heng Yeoh³, Charles G. Cranfield^1,2, Victoria Timchenko³, Bruce A. Cornell^2,4, Stella M. Valenzuela^1,2,5

¹School of Life Sciences, University of Technology Sydney, ²ARC Research Hub for Integrated Devices for End-user Analysis at Low-levels (IDEAL), Faculty of Science, University of Technology Sydney, ³School of Mechanical and Manufacturing Engineering, The University of New South Wales, ⁴Surgical Diagnostics Pty Ltd., ⁵Institute for Biomedical Materials and Devices, University of Technology Sydney

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Biology

Temporary Translocation of Entire Mistletoe Plants to Understand the Mechanistic Basis of Animal Foraging Decisions

Melinda Cook¹, Andy Leigh¹, David M. Watson²

¹School of Life Sciences, University of Technology Sydney, ²Gulbali Institute, School of Agricultural, Environmental, and Veterinary Science, Charles Sturt University

This work outlines a simple experimental procedure to quantify behavioral drivers of foraging decisions in free-living animals, temporarily relocating mistletoe plants to novel locations and measuring visitation rates.

Chemistry

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Sercan Pravadali-Cekic¹, Jake P. Violi¹, Simon M. Mitrovic², Kenneth J. Rodgers², David Bishop¹

¹Hyphenated Mass Spectrometry Laboratory (HyMaS), University of Technology Sydney, ²School of Life Sciences, University of Technology Sydney

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Biology

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples

Jennifer L. Matthews¹, Natasha Bartels¹, Sheik Nadeem Elahee Doomun², Simon K. Davy³, David P. De Souza²

¹Climate Change Cluster (C3), University of Technology Sydney, ²Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, ³School of Biological Sciences, Victoria University of Wellington

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.