Automated systems for cell transfection
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(a) Image of RTCM created using the Nanoprint MicroArray System located at MCN. (b) Close-up of Array Spots. Protein vectors are tagged with GFP and appear green. HeLa Cell Nuclei are labelled with DAPI and appear blue. DNA is cy3 labelled and appears red.
March 2012
Research areas that involve time consuming and repetitive processes may benefit by adopting new experimentation methods, which involve efficient screening. Incorporating nanosystems into these experiments can also provide useful insight into the interaction of nanomaterials within the biological environment, gene expression, targeted cell delivery and encapsulation.
Michael Nastasie, a researcher from Monash University is working in collaboration with MCN’s Varsha Lal to design a novel process capable of conducting a series of simultaneous nano-experiments within an automated array system. This will allow them to observe the transfection of numerous known DNA constructs into specific mammalian cell populations.
Transfection is the process of deliberately introducing nucleic acids into cells. Using the MCN’s MicroArray system, the team can perform transfection experiments simultaneously using the high throughput capabilities of the microarray system. According to Michael, “the microarray system available at the MCN will be crucial in the ability to simultaneously test numerous different transfection solutions, on a large variety of surfaces, under controlled humidified conditions, with minimal user interaction.” Since an array can contain tens of thousands of probes, the automation and concurrent nature accomplished by this MicroArray system drastically improves the efficiency of experimentation and accuracy of results and gives researchers the power to test large quantities of cells in concurrent experiments.
Synthesising high quality enzymes
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Microfluidic device designs for the production of high quality enzymes
March 2012
Enzymes, organic catalysts capable of facilitating physicochemical and biological reactions, have captured the attention of industry and the scientific community alike. In nature, enzymes are used to catalyse and break down proteins. New methods of synthetic production have vastly broadened the applications of such enzymes to include food processing, textile and paper production, biological detergents and farming, medical and therapeutic processes, fuel production and sustainability practices.
According to Florian Lapierre from CSIRO, one of the major challenges of synthetic enzyme production lies not in the overall yield size, but achieving a product that is of consistently high quality. Using the soft lithography capabilities within the MCN’s cleanroom facility, a micro droplet sorting device has been fabricated and is currently undergoing extensive testing. The prototype is capable of generating amino acid emulsions, synthesising proteins via electrically induced coalescence, detection and subsequent sorting of high quality enzymes. The result is a purified sample, which can then be used for catalytic processes or stored for later use.
Identifying key materials for chemical sensors
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(a) Set-up for combinatorial sputter approach of metal oxide thin films, (b, c) cross- sectional view of the fabricated sensors.
February 2012
This project looks at the creaton of a rapid, convenient, and effective solution for discovering multicompositional materials with the best performance from a large compositional landscape.
To achieve this, MCN’s Dr. Sasi Kandasamy collaborated with Dr. Adrian Trinchi and Dr. Tim Muster from the Centre for Materials Science and Engineering and CSIRO.
In designing chemical sensors, combinatorial approaches facilitate with relative ease the discovery of new material compositions with optimised chemical sensing properties, particularly when combined with high-throughput analysis. This in turn allows for libraries of materials that have high sensitivity and selectivity towards specific analyte chemical species to be established.
In this collaborative initiative, the project uses an array of approximately 12 x 12 sensors, fabricated on a 4-inch silicon wafer and tested for their hydrogen gas sensing characteristics using high-throughput impedance spectroscopy. Metal oxide thin films with different compositions of titanium, tungsten and molybdenum were deposited using reactive sputtering at different sites on a single substrate by using masks to define the film deposition sites. The different signal fingerprints of the sensors with different concentrations of these elements successfully demonstrates the validity of these combinatorial chemical sensor design.
50nm biosensors to detect antigens
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a) CAD file outlining the position of the alignment markers with respect to the single device; b) SEM images of the device with markers, and a close up on the device;
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c) SEM image of the nanowire, less then 35nm wide, placed in between the electrodes very accurately. d) SEM image of the device dry-etched to the desired depth.
February 2012
This project aims to develop a set of processes to fabricate a biological nano-sensor which is sensitive to a specific type of biological antigen. To achieve this, MCN’s Dr. Matteo Altissimo and Thanh Nguyen from the University of Melbourne fabricated a set of gold contacts and gold alignment markers on a silicon wafer by means of Electron Beam Lithography (EBL), metal deposition and lift-off. A nanowire was then overlayed on existing gold contacts using the automatic alignment routines of the Vistec EBPG5000plus EBL tool. The negative resist used in this step was then used as a mask for dry etching of the underlying silicon, to a depth of 50nm.
The nanowire surface was then be functionalised by specific antibodies, which selectively bound to their complementary antigens. The binding of antigens could then be detected using electrical measurements.
Interestingly, the EBL tool used only four markers to align all of the exposed nanowires. Despite the fact that the markers were on average more than 20mm away from the target position of the nanowire, the accuracy of the overlay is better than 10nm. This is equivalent to hitting a target 20 km away within an accuracy of 10mm, after measuring the position of 4 template markers.
Predicting artery plaque rupture
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Sphere (left) attached to the cantilever tip (right).
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AFM deflection image of cancerous (left) and benign breast cells (right).
February 2012
Due to lipid build up in artery walls, plaques form that can rupture and cause significant health problems for patients. Researchers at the Baker institute have worked together with MCN to study and predict the likelihood of plaque formation and rupture due to Atherosclerosis in an attempt to prevent it.
Atherosclerosis occurs when lipid steaks infiltrate and build-up in the artery walls, creating a plaque as a result. These plaques can be divided in two distinct types: those that rupture (vulnerable) and those that are less likely to rupture (stable).
In this project, the Nanowizard II AFM was used to determine the elastic properties of the vessels and plaque as well as the topography of the plaque surface. The first results were directly implemented in computational fluid dynamics software. Studying the surface topography then led to the development of models for the boundary conditions along the artery walls. This work will employ a new model in mice, to be used for predicting the formation and properties of vulnerable plaques.
A difficulty in the present approach is working with wet and soft samples. In particular, working with soft samples implies a specific set up for modified cantilevers, in which a sphere is bound to the AFM cantilever (see image above). Tests will be conducted using breast cancer cells to improve this methodology and optimise the conditions for cantilevers. Cantilever optimisation experiments will utilise the cleanroom facility at MCN.
Liquitab, not a hard pill to swallow
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The liquitab will potentially assist millions in taking their daily medications.
February 2012
The days of taking costly liquid antibiotics will soon be over for people suffering dysphagia - discomfort when swallowing pills. Liquitab Systems Ltd. has engaged with MCN to develop a unique technology, capable of grinding pills into a palatable liquid
Utilising facilities at MCN, Liquitab have developed a unique technology capable of crushing and grinding commercially pressed-pill medications into a palatable liquid. The technology harnesses high frequency ultrasonic vibrations to grind pressed-pill medicines and is aimed at assisting those who have difficulty swallowing conventional tablets.
The Liquitab tablet-crushing technology involves the deformation of a metal ring embedded with a removable non-reactive cup. The pressed pill is placed into the cup and ground using the resonant frequency omitted by a transducer. The result is a powdered medication that can be mixed with water to assist with administration.
Professor James Friend applied his expertise and knowledge of ultrasonics to enhance performance of the device. According to Professor Friend, “Tweaking and refining the design of the connecting arm, and adjusting the resonant frequency omitted by the transducer,” produced a far more efficient delivery of ultrasonic vibration. Upon completion of the simulation, design and testing phases, the transition time from solid to tablet powder was reduced from 6 minutes to approximately 1 minute.
This collaboration was facilitated by Grey Innovation and further highlights how the STIUP program can facilitate innovation between industry and academia.
Enhancing Victorian mussel farms’ profitability
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1: 3-dimensional polymer structure for research in mussel farming
February 2012
Utilising the Objet 3D printer for rapid prototyping, researchers at MCN in a collaboration with CSIRO and James Cook University, designed a 3D polymer structure to improve mussel spat capture and enhance the settlement of larvae to ultimately improve the profitability of Victorian mussel farms.
The project aimed to fabricate surface textures that enhance the settlement of mussel larvae, to improve capture of wild mussel spat and therefore profitability. Dr. Andrew Poole from CSIRO and Professor Rocky de Nys from James Cook University utilised the Eden 260V 3-dimensional printing system at MCN to create a fast, clean and accurate prototype of a polymer micro-structured surface. This flexibility in the rapid protoyping cycle allowed the collaborators to immediately change the texture design and produce new polymer surfaces to suit their research requirements. The image above depicts an image of the 400μm groove with 800μm spacing polymer structure that was produced on the 3-dimensional printing system. These surfaces are currently being used for trials at Queenscliff Mussel Hatchery (Department of Primary Industries).
Nanoscale study of biological cells
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SEM image of a single cell showing the FIB milled areas. The top row shows 1x1 μm2 areas milled at 5 kV, and the bottom row shows 1x1 μm2 areas milled at 30 kV.
February 2012
Focused Ion Beam Scanning Electron Microscopes (FIB-SEM) are able to mill away at layers of a material in a very small, very uniform way. This is enabling the study of biological cells on a nanoscale as researchers at MCN and Monash University are utilising the FIB-SEM as well as protein nanoprobes to study protein assemblies and their place within cells. This project aims to find the optimum FIB milling conditions to mill away a very small, uniform thickness (10nm - 50nm) of a single biological cell. The ability to selectively remove such a thin, uniform layer from a single cell, coupled with protein nanoprobes, will be able to describe protein assemblies and their associated locations inside a cell, providing valuable information for drug delivery and targeted therapies.
As an initial stage, the team are characterising the FIB milling rate of a single biological cell at different beam conditions. Initial experimental results indicate that the FIB milling rate at 30kV is approximately 2μm3/nC, while the milling rate at 5kV is approximately 0.6 μm3/nC, indicating that it may be more suitable to use the FIB at 5kV for delicate milling of cells.
Further experiments are currently underway to validate initial findings and to characterise the cell morphology before and after FIB milling to determine the damage caused by the FIB milling process.
Creating corrosion-proof materials
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February 2012
Corrosion is an every-day problem that shortens the life of machinery, buildings, cars and infrastructure. CSIRO have engaged with MCN to develop oxide coated materials to prevent corrosion and ultimately, rust proof the future’s metals. The project looks at measuring surface charge of coated material for anti-corrosion.
The High Throughput Inorganic Coatings research team at CSIRO has been developing novel two, three and four layered coatings of various metal oxide compounds. These oxide formulations have been deposited from positions to yield unique combinations of layered gradient coatings. The coatings are being developed for use in sensing and in anti-corrosion structural protection. Of particular importance in developing anti-corrosion oxides is measuring the surface charge of the coating versus pH; the Surpass Zeta Potential analyzer at the MCN was utilised to achieve this. The investigation showed the surface charge is strongly dependent on the coating thickness, allowing the researchers to tune the thickness to obtain zero charge on the surface in elimination of corrosion.
Rapid point of care sensor for infectious disease discrimination
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SEM of an array of pyramid wells on a silicon master.
October 2011
Together with Biodetectors Pty Ltd, MCN is working on the rapid point of care sensor for infectious disease discrimination. This technology is useful in the fields of genetic screening, diagnostics, drug delivery and protein analysis
Despite their popularity, microarrays (an ordered array of microscopic elements on a planar substrate) often suffer from slow reaction kinetics and are expensive to fabricate. Bead-based technologies are an attractive alternative to classical microarrays due to the high surface area of the beads, which enables the immobilisation of large numbers of probe molecules leading to increased kinetics and improved signal/noise ratios. This technology allows for fast, specific, high-throughput analysis of target molecules.
MCN’s contribution in this project includes device design, prototype fabrication and optical assay verification. The proposed technology of anisotropically etched pyramidal wells on silicon substrate, and its subsequent use as a template for the PDMS fluorescent bead arrays, is a simple, versatile and inexpensive method for the fabrication of high-density bead arrays. Using the micro/nano fabrication platforms available at the MCN, arrays of pyramidal wells have been patterned and fabricated to allow fluorescent bead molecules to be assembled in them. The image above shows an SEM image of an array of fabricated pyramid wells with a width of 6µm on a silicon master. This work will help demonstrate the potential to create high density arrays with better readout capabilities and superior signal to noise ratio than those achievable with classical microarrays.