Category Archives: publication

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First reversible ‘turn-off’ sensor for Glutathione

6 September 2017:

The first reversible ‘turn-off’ sensor for Glutathione has been reported by CNBP researchers in a paper published in the science journal Biosensors.

The paper is accessible online (open access).

Dr Sabrina Heng notes:

γ-Glutamyl-cysteinyl-glycine (GSH) plays a critical role in maintaining redox homeostasis in biological systems and a decrease in its cellular levels is associated with disease. Many diseases including Parkinson’s, cancer, heart diseases and Alzheimer’s are indicated by a decrease in GSH levels. In this case, a ‘turn on’ sensor would result in reduced fluorescence relative to healthy cells. An important advance would come from the development of a sensor that is measurably turned off by GSH and back on by a lower level of GSH. This would then provide an opportunity to sense reduced GSH levels during the onset of important diseases.

With that in mind we have rationally designed, to the best of our knowledge, the first reversible, reaction-based ‘turn-off’ probe that is suitable for sensing decreasing levels of GSH, a situation known to occur at the onset of various diseases.  We have demonstrated that the sensor can be used to detect changes of intracellular GSH in live HEK 293 cells to provide a potentially regenerable sensor for monitoring lower levels of intracellular GSH as associated with the onset of important diseases.

Journal: Biosensors.

Publication title: A Rationally Designed Reversible ‘Turn-Off’ Sensor for Glutathione.

Authors: Sabrina Heng (pictured), Xiaozhou Zhang, Jinxin Pei and Andrew D. Abell.

Abstract: γ-Glutamyl-cysteinyl-glycine (GSH) plays a critical role in maintaining redox homeostasis in biological systems and a decrease in its cellular levels is associated with diseases. Existing fluorescence-based chemosensors for GSH acts as irreversible reaction-based probes that exhibit a maximum fluorescence (‘turn-on’) once the reaction is complete, regardless of the actual concentration of GSH. A reversible, reaction-based ‘turn-off’ probe (1) is reported here to sense the decreasing levels of GSH, a situation known to occur at the onset of various diseases. The more fluorescent merocyanine (MC) isomer of 1 exists in aqueous solution and this reacts with GSH to induce formation of the ring-closed spiropyran (SP) isomer, with a measurable decrease in absorbance and fluorescence (‘turn-off’). Sensor 1 has good aqueous solubility and shows an excellent selectivity for GSH over other biologically relevant metal ions and aminothiol analytes. The sensor permeates HEK 293 cells and an increase in fluorescence is observed on adding buthionine sulfoximine, an inhibitor of GSH synthesis.

Charge transfer in helical peptides

1 September 2017:

Understanding the electronic properties inherent to peptides is crucial for controlling charge transfer, and precursory to the design and fabrication of bio-inspired next generation electronic components.

However, to achieve this objective one must first be able to predict and control the associated charge transfer mechanisms.

Here CNBP researchers demonstrate for the first time a controllable mechanistic transition in peptides resulting directly from the introduction of a side-bridge.

Journal: RSC Advances.

Publication title: A controllable mechanistic transition of charge transfer in helical peptides: from hopping to superexchange.

Authors: Jingxian Yu (pictured), John R. Horsley and Andrew D. Abell.

For more information, access the paper here.

Exploring small-sized nanoflakes

29 August 2017:

Size-dependent structural and electronic properties of MoSmonolayer nanoflakes, of sizes up to 2nm, have been investigated by CNBP researchers using density-functional theory (DFT). The paper, published in Scientific Reports is accessible online.

Journal: Scientific Reports.

Publication title: A study of size-dependent properties of MoSmonolayer nanoflakes using density-functional theory.

Authors: M. Javaid (pictured), Daniel W. Drumm, Salvy P. Russo & Andrew D. Greentree.

Abstract: Novel physical phenomena emerge in ultra-small sized nanomaterials. We study the limiting small-size-dependent properties of MoS2 monolayer rhombic nanoflakes using density-functional theory on structures of size up to Mo35S70 (1.74 nm). We investigate the structural and electronic properties as functions of the lateral size of the nanoflakes, finding zigzag is the most stable edge configuration, and that increasing size is accompanied by greater stability. We also investigate passivation of the structures to explore realistic settings, finding increased HOMO-LUMO gaps and energetic stability. Understanding the size-dependent properties will inform efforts to engineer electronic structures at the nano-scale.

New technique to aid IVF embryo selection

28 August 2017:

Researchers at the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) have successfully developed an advanced new imaging technique, which can help assess the quality of early-stage embryos.

The research, reported in the journal ‘Human Reproduction’ has the potential to significantly benefit the IVF industry of the future, improving assisted reproduction outcomes for women.

“We use a special type of imaging to show differences in the metabolism and chemical make-up of embryos before they’ve been implanted,” says lead author Dr Mel Sutton-McDowall (pictured).

“This technique can give us an objective measure of which embryo to choose as part of the IVF process.”

This ‘hyperspectral imaging’ measures light that cells naturally produce during their normal activities. The light or ‘autoflorescence’ produced changes according to the chemical reactions or metabolism going on in the cell.

Being able to measure embryo metabolism is viewed by many researchers as one of the most important factors as to whether a particular IVF program will be successful.

However, says Dr Sutton-McDowall, fertility specialists take a largely subjective approach in deciding which embryos should be used.

“Pre-implantation screening of embryos generally takes place under a normal optical microscope. Although it’s quite easy to discern poor embryos (due to differences in uniformity), it is far harder for the clinician to determine objectively, the viability of the other embryos,” she says.

“The challenge is how to choose the single healthiest embryo out of this group to maximise the chances of pregnancy.”

Dr Sutton-McDowall sees the use of hyperspectral imaging as a new tool that can be combined with other diagnostic methods to provide a more accurate and objective embryo viability assessment.

“The benefit of hyperspectral imaging is that it can capture information-rich content of inspected objects. It analyses every pixel in an image for its light intensity at differing wavelengths,” she says

“This lets us drill down and analyse the hyperspectral signature of each individual embryo, looking for known or anomalous characteristics. It lets us discriminate between embryos, but also measuring metabolic differences within individual embryos. We predict that embryos that have cells with homogeneous (uniform) metabolic profiles are the healthier ones.”

To date, this imaging technology has only been tested on cattle embryos but Dr Sutton-McDowall notes that the technique is extremely promising.

“It offers benefits of being a non-invasive imaging approach that provides real-time information to the clinician,” she says.

The paper is accessible online.

Journal: Human Reproduction.

Publication title: Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

Authors: Melanie L. Sutton-McDowall, Martin Gosnell, Ayad G. Anwer, Melissa White, Malcolm Purdey, Andrew D. Abell, Ewa M. Goldys, Jeremy G. Thompson.

Abstract: 

STUDY QUESTION
Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity?

SUMMARY ANSWER
Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy.

WHAT IS KNOWN ALREADY
Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment.

STUDY DESIGN, SIZE, DURATION
The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40–50 embryos per replicate used for the developmental competency experiment, 10–20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21–22 embryos used for the hyperspectral microscopy study.

PARTICIPANTS/MATERIALS, SETTING, METHODS
In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy.

MAIN RESULTS AND THE ROLE OF CHANCE
Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups.

LIMITATIONS, REASONS FOR CAUTION
Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success.

WIDER IMPLICATIONS OF THE FINDINGS
Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer.

Nano-diamond arrays on glass

23 August 2017:

Researchers from CNBP’s RMIT University node (lead author Ashleigh Heffernan), have published a paper demonstrating a directed self-assembly method to position nanodiamonds on glass. The method, allowing for the statistical quantification of fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

The paper is accessible online.

Journal: Scientific Reports.

Publication title: Nanodiamond arrays on glass for quantification and fluorescence characterisation.

Authors: Ashleigh H. Heffernan, Andrew D. Greentree & Brant C. Gibson.

Abstract: Quantifying the variation in emission properties of fluorescent nanodiamonds is important for developing their wide-ranging applicability. Directed self-assembly techniques show promise for positioning nanodiamonds precisely enabling such quantification. Here we show an approach for depositing nanodiamonds in pre-determined arrays which are used to gather statistical information about fluorescent lifetimes. The arrays were created via a layer of photoresist patterned with grids of apertures using electron beam lithography and then drop-cast with nanodiamonds. Electron microscopy revealed a 90% average deposition yield across 3,376 populated array sites, with an average of 20 nanodiamonds per site. Confocal microscopy, optimised for nitrogen vacancy fluorescence collection, revealed a broad distribution of fluorescent lifetimes in agreement with literature. This method for statistically quantifying fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

Want a better camera? Just copy bees!

4 July 2017:

Check out the latest buzz about bees and their extra light-sensing eyes! CNBP CI Prof Andy Greentree is coauthor on a new paper in PNAS, which identifies how the eyes and brains of honeybees work together, to process colour information.

“If we can design technology to mimic the way bees do this, we’ll be able to create better cameras and image-processing systems for drones and robots,” say the researchers in an article on the science news channel ‘The Conversation‘.

 

Enhancement of the NV quantum yield

3 July 2017:

Researchers from CNBP’s RMIT University node (lead author CNBP PhD student Marco Capelli pictured), have had a paper published in the journal ‘Nanoscale’.

The researchers report an enhancement of the nitrogen-vacancy (NV) quantum yield by up to 7% in bulk diamond caused by an external magnetic field.

The paper is accessible online.

Journal: Nanoscale.

Publication title: Magnetic field-induced enhancement of the nitrogen-vacancy fluorescence quantum yield .

Authors: M. Capelli, P. Reineck, D. W. M. Lau, A. Orth, J. Jeske, M. W. Doherty, T. Ohshima, A. D. Greentree and B. C. Gibson.

Abstract: The nitrogen-vacancy (NV) centre in diamond is a unique optical defect that is used in many applications today and methods to enhance its fluorescence brightness are highly sought after. We observed experimentally an enhancement of the NV quantum yield by up to 7% in bulk diamond caused by an external magnetic field relative to the field-free case. This observation is rationalised phenomenologically in terms of a magnetic field dependence of the NV excited state triplet-to-singlet transition rate. The theoretical model is in good qualitative agreement with the experimental results at low excitation intensities. Our results significantly contribute to our fundamental understanding of the photophysical properties of the NV defect in diamond.

Sensors for calcium ion

15 June 2017:

Researchers from CNBP (lead author Dr Sabrina Heng pictured), have just had a paper published, reporting on three new spiropyran-based reversible sensors for calcium ion.

Journal: Sensors and Actuators B: Chemical.

Publication title: Photoswitchable calcium sensor: ‘On’–‘Off’ sensing in cells or with microstructured optical fibers.

Authors: Sabrina Heng, Adrian M. Mak, Roman Kostecki, Xiaozhou Zhang, Jinxin Peia, Daniel B. Stubing, Heike Ebendorff-Heidepriema, Andrew D. Abell.

Abstract: Calcium is a ubiquitous intracellular signaling ion that plays a critical role in the modulation of fundamental cellular processes. A detailed study of these processes requires selective and reversible sensing of Ca2+ and an ability to quantify and monitor concentration changes in a biological setting. Three new, rationally designed, synthesized and photoswitchable spiropyran-based reversible sensors for Ca2+ are reported. Sensor 1a is highly selective for Ca2+ with an improved profile relative to the other two analogues, 1b and 1c. Formation of the merocyanine–Ca2+ complex is proportional to an increase in Ca2+ released from HEK293 cells on stimulation with ionomycin. The photophysical processes surrounding the binding of Ca2+ to compound 1a were further explored using computational methods based on density functional theory (DFT). The ability of sensor 1a to bind Ca2+ and photoswitch reversibly was also characterized using silica suspended-core microstructured optical fiber (SCF). These SCF experiments (with 100 nM Ca2+) represent a first step toward developing photoswitchable, minimally invasive and highly sensitive Ca2+ sensing platforms for use in a biological setting.

The paper is accessible online.

Maximizing particle concentration

28 April 2017:

A new paper from CNBP researchers reports on an improvement to deterministic lateral displacement arrays, which allows for higher particle concentration enhancement. The work has just been published in the journal ‘Biomicrofluidics’ and is accessible online.

Journal: Biomicrofluidics.

Title: Maximizing particle concentration in deterministic lateral displacement arrays.

Authors: Shilun Feng, Alison M. Skelley, Ayad G. Anwer (pictured top left), Guozhen Liu and David W. Inglis.

Abstract: We present an improvement to deterministic lateral displacement arrays, which allows higher particle concentration enhancement. We correct and extend previous equations to a mirror-symmetric boundary. This approach allows particles to be concentrated into a central channel, no wider than the surrounding gaps, thereby maximizing the particle enrichment. The resulting flow patterns were, for the first time, experimentally measured. The performance of the device with hard micro-spheres and cells was investigated. The observed flow patterns show important differences from our model and from an ideal pattern. The 18 μm gap device showed 11-fold enrichment of 7 μm particles and nearly perfect enrichment—of more than 50-fold—for 10 μm particles and Jurkat cells. This work shows a clear path to achieve higher-than-ever particle concentration enhancement in a deterministic microfluidic separation system.

 

Tuning third harmonic light

27 April 2017:

Researchers from CNBP and The Institute of Photonic Technology (lead author Stephen Warren-Smith pictured), have just had a paper published on tuning third harmonic light generated within exposed-core fibres.

Journal: Optics Letters.

Publication title: Nanofilm-induced spectral tuning of third harmonic generation.

Authors: Stephen C. Warren-Smith, Mario Chemnitz, Henrik Schneidewind, Roman Kostecki, Heike Ebendorff-Heidepriem, Tanya M. Monro and Markus A. Schmidt.

Abstract: Intermodal third-harmonic generation using waveguides is an effective frequency conversion process due to the combination of long interaction lengths and strong modal confinement. Here we introduce the concept of tuning the third harmonic phase-matching condition via the use of dielectric nanofilms located on an open waveguide core. We experimentally demonstrate that tantalum oxide nanofilms coated onto the core of an exposed core fiber allow tuning the third harmonic wavelength over 30 nm, as confirmed by qualitative simulations. Due to its generic character, the presented tuning scheme can be applied to any form of exposed core waveguide and will find applications in fields including microscopy, biosensing, and quantum optics.

The paper is accessible online.