10 June 2019
A team of CNBP researchers have published a new paper discussing the design and application of a micro fabricated needle-like probe to measure hydrogen peroxide. This new microfluidic tool has applications for monitoring dynamic chemical reactions in analytical chemistry and biological systems.
Journal: RSC Advances
Publication Title: Microfabricated needle for hydrogen peroxide detection
Authors: Shilun Feng, Sandhya Clement, Yonggang Zhu, Ewa M. Goldys and David W. Inglis
Abstract: A microfabricated needle-like probe has been designed and applied for hydrogen peroxide (H2O2) sampling and detection using a commercial, single-step fluorescent H2O2 assay. In this work, droplets of the assay reagent are generated and sent to the needle tip using a mineral-oil carrier fluid. At the needle tip, the sample is drawn into the device through 100 mm long hydrophilic capillaries by negative pressure. The sampled fluid is immediately merged with the assay droplet and carried away to mix and react, producing a sequence of droplets representing the H2O2 concentration as a function of time. We have characterized the assay fluorescence for small variations in the sample volume. With the calibration, we can calculate the concentration of H2O2 in the sampled liquid from the size and intensity of each merged droplet. This is a microfluidic data-logger system for on-site continuous sampling, controlled reaction, signal storage and on-line quantitative detection. It is a useful tool for monitoring dynamic chemical reactions in analytical chemistry and biological applications.
Key words: Microfluidics, probe, H2O2, analytics chemistry
29 May 2019
Recent publication by CNBP PhD student Mr Yi Li and team at the University of New South Wales explores the challenges and opportunities of working with CRISPR /Cas for multiplex detection
Journal: Trends in Biotechnology
Publication Title: CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?
Authors: Yi Li, Linyang Liu, Guozhen Liu
Abstract: Performing multiplex detection is still an elusive goal for molecular diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics.
CRISPR/Cas; multiplex; biosensing; diagnostics; nucleic acid detection
6 May 2019:
Hemoglobin expression in reproductive cells and the role of hemoglobin on oocyte and early embryo development is the focus of this latest CNBP review paper published in the journal ‘Biology of Reproduction’ (lead author Megan Lim based at the University of Adelaide).
Journal: Biology of Reproduction.
Publication title: Hemoglobin: potential roles in the oocyte and early embryo.
Authors: Megan Lim, Hannah M Brown, Karen L Kind, Jeremy G Thompson, Kylie R Dunning.
Abstract: Hemoglobin (Hb) is commonly known for its capacity to bind and transport oxygen and carbon dioxide in erythroid cells. However, it plays additional roles in cellular function and health due to its capacity to bind other gases including nitric oxide. Further, Hb acts as a potent antioxidant, quenching reactive oxygen species. Despite its potential roles in cellular function, the preponderance of Hb research remains focused on its role in oxygen regulation. There is increasing evidence that Hb expression is more ubiquitous than previously thought, with Hb and its variants found in a myriad of cell types ranging from macrophages to spermatozoa. The majority of non-erythroid cell types that express Hb are situated within hypoxic environments, suggesting Hb may play a role in hypoxia-inducible factor (HIF)-regulated gene expression by controlling the level of oxygen available or as an adaptation to low oxygen providing a mechanism to store oxygen. Oocyte maturation and preimplantation embryo development occur within the low oxygen environments of the antral follicle and oviduct/uterus, respectively. Interestingly, Hb was recently found in human cumulus and granulosa cells and murine cumulus-oocyte complexes (COCs) and preimplantation embryos. Here, we consolidate and analyze the research generated to-date on Hb expression in non-erythroid cells with a particular focus on reproductive cell types. We outline future directions of this research to elucidate the role of Hb during oocyte maturation and preimplantation embryo development and finally, we explore the potential clinical applications and benefits of Hb supplementation during the in vitro culture of gametes and embryos.
1 May 2019:
A molecular imprinted polymer biosensing device (developed on stainless steel) that can successfully detect cytokines has been reported by CNBP researchers. Cytokines are proteins secreted by cells that stimulate surrounding cells into specific action and are important to an organism’s immune responses. The finding was reported in the journal ‘Sensors and Actuators B: Chemical’ with the lead author of the publication being CNBP’s Fei Deng based at UNSW Sydney.
Journal: Sensors and Actuators B: Chemical.
Publication title: Molecularly imprinted polymer-based reusable biosensing device on stainless steel for spatially localized detection of cytokine IL-1β.
Authors: Fei Deng, Ewa M. Goldys, Guozhen Liu.
Abstract: A molecularly imprinted polymer (MIP) based biosensing device on stainless steel (SS) for detection of locally variable concentration of cytokine interleukin-1β (IL-1β) was successfully developed using a sandwich assay scheme. The SS surface was firstly modified with a layer of polydopamine (PDA) followed by the attachment of a layer of poly(ethyleneimine) (PEI) by electrostatic adsorption. Subsequently, the template protein IL-1β was adsorbed on the PEI terminated SS surface due to electrostatic adsorption. A PDA imprinting film was then in-situ synthesized on the surface of the modified SS substrate with incorporated template cytokine. Finally, the template was washed off the SS substrate leaving behind cavities with specific shape and capable of capturing cytokines thus forming a MIP biosensing interface. After exposure to the analyte IL-1β, the MIP biosensing device was incubated with IL-1β detection antibody-modified fluorescent polystyrene beads allowing to determine the amount of captured IL-1β based on fluorescence intensity. The device has been demonstrated to detect IL-1β with low detection limit of 10.2 pg mL−1, and a linear detection range of 25–400 pg mL−1. This MIP biosensing device can be regenerated more than three times with coefficient of variation 2.08%. The device was applied for the detection of IL-1β secreted by rat macrophages, where the good specificity and selectivity were achieved. MIP serves in this device as a superior substitute of antibody with exceptional stability and reusability. The MIP based biosensing technology presented in our work paves a new way for developing a universal and robust sensing platform for the detection of spatially localised small proteins with low physical concentration.
29 April 2019:
A new research publication (lead author CNBP PhD student Fuyuan Zhang) reports on real-time sensing and detection of cytokines using a 3D optical fibre.
Journal: Molecular Systems Design & Engineering.
Publication title: IFN-γ-induced signal-on fluorescence aptasensors: from hybridization chain reaction amplification to 3D optical fiber sensing interface towards a deployable device for cytokine sensing.
Authors: Fuyuan Zhang, Fei Deng, Guo-Jun Liu, Ryan Middleton, David W. Inglis, Ayad Anwer, Shuo Wang and Guozhen Liu.
Abstract: Interferon-gamma (IFN-γ), a proinflammatory cytokine, has been used as an early indicator of multiple infectious diseases or tumors. In order to explore the detection capability of a commonly used anti-IFN-γ aptamer, a simple target induced strand-displacement aptasensing strategy was tested by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor showed the best affinity constant (Kd) of 21.87 ng mL−1. It was found that the strand-displacement aptasensing strategy was impacted by the complementary position and length of the complementary strands. Additionally, the hybridization chain reaction (HCR) amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng mL−1. In order to further explore the sensing platform for spatially localized cytokine detection, the Texas red/BHQ2-based strand-displacement aptasensor was successfully fabricated on the 3D optical fiber surface to achieve a deployable sensing device for monitoring IFN-γ based on the fluorescence spots counting strategy. Finally, the three developed aptasensing strategies (strand-displacement strategy, HCR amplification strategy, 3D optical fiber aptasensor) were applied for detection of IFN-γ secreted by PBMCs with comparable results to those of ELISA. The deployable 3D optical fiber aptasensor with the superior sensitivity is potential to be used for detection of spatially localized IFN-γ in vivo.
26 April 2019:
An advanced new method has been developed by CNBP researchers that may open the door to 3D microscopy in hard-to-reach areas of the human body.
It sees the successful miniaturization of a 3D imaging technique called ‘light field imaging’, taken to extreme new levels, making in-body application possible.
It could find significant application in diagnostic procedures called optical biopsies, where suspicious tissue is investigated during medical endoscopic procedures.
Reported in the journal ‘Science Advances’, project lead of the innovative imaging approach is Dr Antony Orth, Research Fellow at the RMIT University node of the CNBP (pictured).
The paper can be accessed below or read the media release here.
Journal: Science Advances.
Publication title: Optical fiber bundles: Ultra-slim light field imaging probes.
Authors: A. Orth, M. Ploschner, E. R. Wilson, I.S. Maksymov and B. C. Gibson.
Abstract: Optical fiber bundle microendoscopes are widely used for visualizing hard-to-reach areas of the human body. These ultrathin devices often forgo tunable focusing optics because of size constraints and are therefore limited to two-dimensional (2D) imaging modalities. Ideally, microendoscopes would record 3D information for accurate clinical and biological interpretation, without bulky optomechanical parts. Here, we demonstrate that the optical fiber bundles commonly used in microendoscopy are inherently sensitive to depth information. We use the mode structure within fiber bundle cores to extract the spatio-angular description of captured light rays—the light field—enabling digital refocusing, stereo visualization, and surface and depth mapping of microscopic scenes at the distal fiber tip. Our work opens a route for minimally invasive clinical microendoscopy using standard bare fiber bundle probes. Unlike coherent 3D multimode fiber imaging techniques, our incoherent approach is single shot and resilient to fiber bending, making it attractive for clinical adoption.
Below – Modal structure in optical fiber bundles captures light field information. Credit Antony Orth, RMIT University.
17 April 2019:
One of the biggest challenges associated with exposed core glass optical fiber-based sensing is the availability of techniques that can be used to generate reproducible, homogeneous and stable surface coating. CNBP scientists report a one step, solvent free method for surface functionalization of exposed core glass optical fiber that allows for the binding of fluorophore-of-choice for metal ion sensing.
Lead author of the paper, published in the journal ‘Sensors’, is CNBP researcher Dr Akash Bachhuka based at the University of Adelaide.
Publication Title: Surface Functionalization of Exposed Core Glass Optical Fiber for Metal Ion Sensing;
Authors: Akash Bachhuka, Sabrina Heng, Krasimir Vasilev, Roman Kostecki, Andrew Abell and Heike Ebendorff-Heidepriem
One of the biggest challenges associated with exposed core glass optical fiber-based sensing is the availability of techniques that can be used to generate reproducible, homogeneous and stable surface coating. We report a one step, solvent free method for surface functionalization of exposed core glass optical fiber that allows achieving binding of fluorophore of choice for metal ion sensing. The plasma polymerization-based method yielded a homogeneous, reproducible and stable coating, enabling high sensitivity aluminium ion sensing. The sensing platform reported in this manuscript is versatile and can be used to bind different sensing molecules opening new avenues for optical fiber-based sensing.
5 April 2019:
A new review article by CNBP PhD student Rachit Bansal and CNBP Associate Investigators (Anwar Sunna, Andrew Care and Tiffany Walsh) reports on experimental tools to study the binding mechanism of synthetic peptides to solid materials. The review provides insights into the role of these peptides as molecular building blocks for nanobiotechnology.
Journal: New Biotechnology.
Publication title: Experimental and theoretical tools to elucidate the binding mechanisms of solid-binding peptides.
Authors: Rachit Bansal, Andrew Care, Megan S. Lord, Tiffany R. Walsh, Anwar Sunna.
Abstract: The interactions between biomolecules and solid surfaces play an important role in designing new materials and applications which mimic nature. Recently, solid-binding peptides (SBPs) have emerged as potential molecular building blocks in nanobiotechnology. SBPs exhibit high selectivity and binding affinity towards a wide range of inorganic and organic materials. Although these peptides have been widely used in various applications, there is a need to understand the interaction mechanism between the peptide and its material substrate, which is challenging both experimentally and theoretically. This review describes the main characterisation techniques currently available to study SBP-surface interactions and their contribution to gain a better insight for designing new peptides for tailored binding.
29 March 2019:
Liquid-metal nanoparticles can focus ultraviolet light at the nanoscale. Read more in a publication reporting on the UV plasmonic properties of colloidal gallium-indium particles (lead author CNBP Associate Investigator Dr Philipp Reineck, RMIT University).
Journal: Scientific Reports.
Publication title: UV plasmonic properties of colloidal liquid-metal eutectic gallium-indium alloy nanoparticles.
Authors: Philipp Reineck, Yiliang Lin, Brant C. Gibson, Michael D. Dickey, Andrew D. Greentree, Ivan S. Maksymov.
Abstract: Nanoparticles made of non-noble metals such as gallium have recently attracted significant attention due to promising applications in UV plasmonics. to date, experiments have mostly focused on solid and liquid pure gallium particles immobilized on solid substrates. However, for many applications, colloidal liquid-metal nanoparticle solutions are vital. Here, we experimentally demonstrate strong UV plasmonic resonances of eutectic gallium-indium (eGaIn) liquid-metal alloy nanoparticles suspended in ethanol. We rationalise experimental results through a theoretical model based on Mie theory. our results contribute to the understanding of UV plasmon resonances in colloidal liquid-metal eGaIn nanoparticle suspensions. they will also enable further research into emerging applications of UV plasmonics in biomedical imaging, sensing, stretchable electronics, photoacoustics, and electrochemistry.
21 March 2019:
A new time-gated microscopy approach has been reported by CNBP researchers that will help neurobiologists better visualize neurokine signaling (and other) molecules in cells or tissue samples. Lead author of the publication is CNBP researcher Dr Lindsay Parker, Macquarie University.
Journal: Journal of Neuroinflammation.
Publication title: Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.
Authors: Lindsay M. Parker, Nima Sayyadi, Vasiliki Staikopoulos, Ashish Shrestha, Mark R. Hutchinson and Nicolle H. Packer.
Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases.
In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples.
Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model.
Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.