21 November 2017:
A new hybrid sensor combining an organic fluorescent probe bound to a nanodiamond has been developed by CNBP researchers (lead author Dr Malcolm Purdey pictured). Able to detect hydrogen peroxide, the sensor is non-toxic and is also highly photostable.
Journal: Scientific Reports.
Publication title: An organic fluorophore-nanodiamond hybrid sensor for photostable imaging and orthogonal, on-demand biosensing.
Authors: Malcolm S. Purdey, Patrick K. Capon, Benjamin J. Pullen, Philipp Reineck, Nisha Schwarz, Peter J. Psaltis, Stephen J. Nicholls, Brant C. Gibson & Andrew D. Abell.
Abstract: Organic fluorescent probes are widely used to detect key biomolecules; however, they often lack the photostability required for extended intracellular imaging. Here we report a new hybrid nanomaterial (peroxynanosensor, PNS), consisting of an organic fluorescent probe bound to a nanodiamond, that overcomes this limitation to allow concurrent and extended cell-based imaging of the nanodiamond and ratiometric detection of hydrogen peroxide. Far-red fluorescence of the nanodiamond offers continuous monitoring without photobleaching, while the green fluorescence of the organic fluorescent probe attached to the nanodiamond surface detects hydrogen peroxide on demand. PNS detects basal production of hydrogen peroxide within M1 polarised macrophages and does not affect macrophage growth during prolonged co-incubation. This nanosensor can be used for extended bio-imaging not previously possible with an organic fluorescent probe, and is spectrally compatible with both Hoechst 33342 and MitoTracker Orange stains for hyperspectral imaging.
17 November 2017:
A new CNBP paper “Statistically strong label-free quantitative identification of native fluorophores in a biological sample,” by Saabah B. Mahbub (first author pictured), Martin Plöschner, Martin E. Gosnell, Ayad G. Anwer and Ewa M. Goldys has just been published in Scientific Reports and is available online.
This work addresses a genuine shortage of methods for real-time continuous monitoring of biochemistry of cells and tissues, especially live cells. Saabah Mahbub and team developed an automated and unbiased unmixing methodology to non-invasively detect the presence and spatial distributions of endogenous fluorophores in retina cells. The method was validated on artificial images, where the addition of a varying known level of noise has allowed to quantify the accuracy of spectral unmixing.
With its capability for high throughput, automation and embedded compatibility with statistical analysis this work will contribute to improved quantification and objectivity in biomedical research.
16 November 2017:
CNBP and Macquarie University PhD candidate Shilun Feng is first author on a new paper exploring a ‘membrane-on-a-chip’ device. The technology has the potential to form an integral part of a new type of microneedle that would be able to transport tiny and precise amounts of fluid/medication within the body.
Publication title: Microfluidic Droplet Extraction by Hydrophilic Membrane.
Authors: Shilun Feng, Micheal N. Nguyen, and David W. Inglis.
Abstract: Droplet-based microfluidics are capable of transporting very small amounts of fluid over long distances. This characteristic may be applied to conventional fluid delivery using needles if droplets can be reliably expelled from a microfluidic channel. In this paper, we demonstrate a system for the extraction of water droplets from an oil-phase in a polymer microfluidic device. A hydrophilic membrane with a strong preference for water over oil is integrated into a droplet microfluidic system and observed to allow the passage of the transported aqueous phase droplets while blocking the continuous phase. The oil breakthrough pressure of the membrane was observed to be 250 ± 20 kPa, a much greater pressure than anywhere within the microfluidic channel, thereby eliminating the possibility that oil will leak from the microchannel, a critical parameter if droplet transport is to be used in needle-based drug delivery.
7 November 2017:
CNBP researchers Dr Daniel Drumm (lead author pictured) and Prof Andrew Greentree, both at RMIT University, have analysed microscopy in the contexts of Rényi-Ulam games and half-lies, developing a new family of heuristics. Their research is reported in the journal ‘Scientific Reports.’
Journal: Scientific Reports.
Publication title: Microscopy as a statistical, Rényi-Ulam, half-lie game: a new heuristic search strategy to accelerate imaging.
Authors: Daniel W. Drumm & Andrew D. Greentree.
Abstract: Finding a fluorescent target in a biological environment is a common and pressing microscopy problem. This task is formally analogous to the canonical search problem. In ideal (noise-free, truthful) search problems, the well-known binary search is optimal. The case of half-lies, where one of two responses to a search query may be deceptive, introduces a richer, Rényi-Ulam problem and is particularly relevant to practical microscopy. We analyse microscopy in the contexts of Rényi-Ulam games and half-lies, developing a new family of heuristics. We show the cost of insisting on verification by positive result in search algorithms; for the zero-half-lie case bisectioning with verification incurs a 50% penalty in the average number of queries required. The optimal partitioning of search spaces directly following verification in the presence of random half-lies is determined. Trisectioning with verification is shown to be the most efficient heuristic of the family in a majority of cases.
6 November 2017:
New research from CNBP scientists reports on a cytokine sensor – fabricated on the surface of an optical fibre. Cytokines are molecules that play a critical role in cellular response to infection, inflammation, trauma and disease. Lead author on the paper, published in the journal ‘Biosensors and Bioelectronics’, is Centre PhD student Kaixin Zhang who is based at Macquarie University.
Journal: Biosensors and Bioelectronics.
Publication title: Robust immunosensing system based on biotinstreptavidin coupling for spatially localized femtogram mL−1 level detection of interleukin-6.
Authors: Kaixin Zhang, Guozhen Liu, Ewa M. Goldys.
Abstract: Detection of a very low amount of cytokines such as interleukin-6 (IL-6) in clinical fluids is important in biomedical research and clinical applications. Here, we demonstrate spatially-localised ultrasensitive (femtogram mL−1) level detection of IL-6 in serum and in cell culture media. Our approach is based on a sandwich immunosensor fabricated on the surface of an optical fibre. Firstly, the biotinylated IL-6 capture antibody was immobilized on the fibre surface by biotin-streptavidin coupling. Then the fabricated fibre was used for capturing IL-6 followed by exposure to detection antibody which was labeled with the fluorescent magnetic nanoparticles to report the signal. A linear relationship between IL-6 concentration and the fluorescence signal was obtained in the range from 0.4 pg mL−1 to 400 pg mL−1 of IL-6, with the limit of detection down to 0.1 pg mL−1. In addition, this optical fibre sensor was successfully applied for the localized detection of IL-6 with the spatial resolution of 200 µm and a sample volume of 1 μL. Finally, the performance of the fibre sensor was demonstrated by detection of IL-6 secreted by BV-2 cells with comparable performance of the conventional enzyme-linked immunosorbent assay (ELISA).
31 October 2017:
Surface chemistry is vital for nanodiamond fluorescence, reports a new paper published by CNBP researchers (lead author Dr Philipp Reineck pictured). The paper was published in the journal ‘ACS Nano’ and is available online.
Journal: ACS Nano.
Publication title: Effect of Surface Chemistry on the Fluorescence of Detonation Nanodiamonds.
Authors: Philipp Reineck, Desmond W. M. Lau, Emma R. Wilson, Kate Fox, Matthew R. Field, Cholaphan Deeleepojananan, Vadym N. Mochalin, and Brant C. Gibson.
Abstract: Detonation nanodiamonds (DNDs) have unique physical and chemical properties that make them invaluable in many applications. However, DNDs are generally assumed to show weak fluorescence, if any, unless chemically modified with organic molecules. We demonstrate that detonation nanodiamonds exhibit significant and excitation-wavelength-dependent fluorescence from the visible to the near-infrared spectral region above 800 nm, even without the engraftment of organic molecules to their surfaces. We show that this fluorescence depends on the surface functionality of the DND particles. The investigated functionalized DNDs, produced from the same purified DND as well as the as-received polyfunctional starting material, are hydrogen, hydroxyl, carboxyl, ethylenediamine, and octadecylamine-terminated. All DNDs are investigated in solution and on a silicon wafer substrate and compared to fluorescent high-pressure high-temperature nanodiamonds. The brightest fluorescence is observed from octadecylamine-functionalized particles and is more than 100 times brighter than the least fluorescent particles, carboxylated DNDs. The majority of photons emitted by all particle types likely originates from non-diamond carbon. However, we locally find bright and photostable fluorescence from nitrogen-vacancy centers in diamond in hydrogenated, hydroxylated, and carboxylated detonation nanodiamonds. Our results contribute to understanding the effects of surface chemistry on the fluorescence of DNDs and enable the exploration of the fluorescent properties of DNDs for applications in theranostics as nontoxic fluorescent labels, sensors, nanoscale tracers, and many others where chemically stable and brightly fluorescent nanoparticles with tailorable surface chemistry are needed.
A new paper from CNBP researchers (lead author Shilun Feng pictured) reports on the development of a microfluidic needle-like device that can extract and deliver nanoliter samples.
The paper, published in ‘Applied Physics Letters’ is accessible online.
Journal: Applied Physics Letters.
Publication title: A microfluidic needle for sampling and delivery of chemical signals by segmented flows.
Authors: Shilun Feng, Guozhen Liu, Lianmei Jiang, Yonggang Zhu, Ewa M. Goldys, and David W. Inglis.
Abstract: We have developed a microfluidic needle-like device that can extract and deliver nanoliter samples. The device consists of a T-junction to form segmented flows, parallel channels to and from the needle tip, and seven hydrophilic capillaries at the tip that form a phase-extraction region. The main microchannel is hydrophobic and carries segmented flows of water-in-oil. The hydrophilic capillaries transport the aqueous phase with a nearly zero pressure gradient but require a pressure gradient of 19 kPa for mineral oil to invade and flow through. Using this device, we demonstrate the delivery of nanoliter droplets and demonstrate sampling through the formation of droplets at the tip of our device. During sampling, we recorded the fluorescence intensities of the droplets formed at the tip while varying the concentration of dye outside the tip. We measured a chemical signal response time of approximately 3 s. The linear relationship between the recorded fluorescence intensity of samples and the external dye concentration (10–40 μg/ml) indicates that this device is capable of performing quantitative, real-time measurements of rapidly varying chemical signals.
26 October 2017:
A new paper featuring CNBP researchers (lead author Sandhya Clement pictured top left), demonstrates a simple, non-destructive method suitable for rapid evaluation of nanoparticles in colloidal suspension.
The paper, published in Nanotechnology is accessible online.
Publication title: Quantification of nanoparticle concentration in colloidal suspensions by a non-destructive optical method.
Authors: Sandhya Clement, Brint Gardner, Wan Aizuddin W Razali, Victoria A Coleman, Åsa K Jämting, Heather J Catchpoole, Ewa M Goldys, Jan Herrmann and Andrei Zvyagin.
Abstract: The estimation of nanoparticle number concentration in colloidal suspensions is a prerequisite in many procedures, and in particular in multi-stage, low-yield reactions. Here, we describe a rapid, non-destructive method based on optical extinction and dynamic light scattering (DLS), which combines measurements using common bench-top instrumentation with a numerical algorithm to calculate the particle size distribution (PSD) and concentration. These quantities were derived from Mie theory applied to measurements of the optical extinction spectrum of homogeneous, non-absorbing nanoparticles, and the relative PSD of a colloidal suspension. The work presents
an approach to account for PSDs achieved by DLS which, due to the underlying model, may not be representative of the true sample PSD. The presented approach estimates the absolute particle number concentration of samples with mono-, bi-modal and broad size distributions with <50% precision. This provides a convenient and practical solution for number concentration estimation required during many applications of colloidal nanomaterials.
16 October 2017:
A tuneable and programmable integrating sphere light source for wide-field fluorescent microscopy imaging, employing nine light-emitting diodes (LEDs), has been successfully demonstrated and reported by CNBP researchers in a new paper (lead author Aziz ul Rehman pictured).
The paper, published in ‘Photodiagnosis and Photodynamic Therapy’ is accessible online.
Journal: Photodiagnosis and Photodynamic Therapy.
Publication title: Programmable LED-Based Integrating Sphere Light Source for Wide-Field Fluorescence Microscopy.
Authors: Aziz ul Rehman, Ayad G.Anwer, Ewa M.Goldys.
Abstract: Wide-field fluorescence microscopy commonly uses a mercury lamp, which has limited spectral capabilities. We designed and built a programmable integrating sphere light (PISL) source which consists of nine LEDs, light-collecting optics, a commercially available integrating sphere and a baffle. The PISL source is tuneable in the range 365–490 nm with a uniform spatial profile and a sufficient power at the objective to carry out spectral imaging. We retrofitted a standard fluorescence inverted microscope DM IRB (Leica) with a PISL source by mounting it together with a highly sensitive low- noise CMOS camera. The capabilities of the setup have been demonstrated by carrying out multispectral autofluorescence imaging of live BV2 cells.
12 October 2017:
Researchers from the CNBP have released a new paper that examines the use of nanorubies for targeted bio-imaging activity. The work (lead author Varun Sreenivasan pictured) is trans-disciplinary in nature, drawing on the Centre’s collective knowledge in physics, pharmacology, chemistry, material science and embryology. The paper, published in ACS Applied Materials and Interfaces is accessible online.
Journal: ACS Applied Materials and Interfaces.
Publication title: Development of Bright and Biocompatible Nanoruby and its Application to Background-free Time-gated Imaging of G-protein Coupled Receptors.
Authors: Varun K. A. Sreenivasan, Wan Aizuddin W Razali, Kai Zhang, Rashmi R Pillai, Avishkar Saini, Denitza Denkova, Marina Santiago, Hannah Brown, Jeremy Thompson, Mark Connor, Ewa M. Goldys, and Andrei V Zvyagin.
Abstract: At the forefront of development of fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially towards demanding applications of single molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (3.7 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high volume production and molecularly-specific targeting. The present work encompasses large scale production of colloidally stable nanoruby particles, demonstration of their biofunctionality and negligible cytotoxicity, as well as validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to µ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single particle sensitivity.