Enzyme Kinetics Analysis: An online tool for analyzing enzyme initial rate data and teaching enzyme kinetics.

Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time-consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web-tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis-Menten, Hill, simple reversible inhibition models, ternary-complex, and ping-pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g., K M or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements.

Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 µm width, and 500 µm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 2: Data extraction, processing and analysis.

The rate at which fluorescently-labeled biomolecules, that are flowing at a constant speed in a microfluidic channel, diffuse into an adjacent buffer stream can be used to calculate the diffusion coefficient of the molecule, which then gives a measure of its size. Experimentally, determining the rate of diffusion involves capturing concentration gradients in fluorescence microscopy images at different distances along the length of the microfluidic channel, where distance corresponds to residence time, based on the flow velocity. The preceding chapter in this journal covered the development of the experimental setup, including information about the microscope camera detection systems used to acquire fluorescence microscopy data. In order to calculate diffusion coefficients from fluorescence microscopy images, intensity data are extracted from the images and then appropriate methods of processing and analyzing the data, including the mathematical models used for fitting, are applied to the extracted data. This chapter begins with a brief overview of digital imaging and analysis principles, before introducing custom software for extracting the intensity data from the fluorescence microscopy images. Subsequently, methods and explanations for performing the necessary corrections and appropriate scaling of the data are provided. Finally, the mathematics of one-dimensional molecular diffusion is described, and analytical approaches to obtaining the diffusion coefficient from the fluorescence intensity profiles are discussed and compared.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 1: Chip development, system operation and measurement setup.

The recent advent of laminar flow-based microfluidic systems for molecular interaction analysis has enabled transformative new profiling of proteins in regards to their structure, disordering, complex formation and interactions in general. Based on the diffusive transport of molecules perpendicular to the direction of laminar flow in a microfluidic channel, systems of this type promise continuous-flow, high-throughput screening of complex, multi-molecule interactions, while remaining tolerant to heterogeneous mixtures. Using common microfluidic device processing, the technology provides unique opportunities, as well as device design and experimental challenges, for integrative sample handling approaches that can investigate biomolecular interaction events in complex samples with readily available laboratory equipment. In this first chapter of a two-part series, we introduce system design and experimental setup requirements for a typical laminar flow-based microfluidic system for molecular interaction analysis in the form of what we call the 'LaMInA system' (Laminar flow-based Molecular Interaction Analysis system). We provide microfluidic device development advice on choice of device material, device design, including impact of channel geometry on the signal acquisition, and on design limitations and possible post-fabrication treatments to redress these. Finally. we cover aspects of fluidic actuation, such as selecting, measuring and controlling the flow rate appropriately, and provide a guide to possible fluorescent labels for proteins, as well as options for the fluorescence detection hardware, all in the context of assisting the reader in developing their own laminar flow-based experimental setup for biomolecular interaction analysis.

Application of sequential cyclic compression on cancer cells in a flexible microdevice.

Mechanical forces shape physiological structure and function within cell and tissue microenvironments, during which cells strive to restore their shape or develop an adaptive mechanism to maintain cell integrity depending on strength and type of the mechanical loading. While some cells are shown to experience permanent plastic deformation after a repetitive mechanical tensile loading and unloading, the impact of such repetitive compression on deformation of cells is yet to be understood. As such, the ability to apply cyclic compression is crucial for any experimental setup aimed at the study of mechanical compression taking place in cell and tissue microenvironments. Here, we demonstrate such cyclic compression using a microfluidic compression platform on live cell actin in SKOV-3 ovarian cancer cells. Live imaging of the actin cytoskeleton dynamics of the compressed cells was performed for varying pressures applied sequentially in ascending order during cell compression. Additionally, recovery of the compressed cells was investigated by capturing actin cytoskeleton and nuclei profiles of the cells at zero time and 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. As demonstrated in this work, the observation of morphological, cytoskeletal and nuclear differences in compressed and non-compressed cells, with controlled micro-scale mechanical cell compression and recovery and using live-cell imaging, fluorescent tagging and end point assays, can give insights into the mechanics of cancer cells.

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta.

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.

On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the 'holy grail' for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.

Microdevice-based mechanical compression on living cells.

Compressive stress enables the investigation of a range of cellular processes in which forces play an important role, such as cell growth, differentiation, migration, and invasion. Such solid stress can be introduced externally to study cell response and to mechanically induce changes in cell morphology and behavior by static or dynamic compression. Microfluidics is a useful tool for this, allowing one to mimic in vivo microenvironments in on-chip culture systems where force application can be controlled spatially and temporally. Here, we review the mechanical compression applications on cells with a broad focus on studies using microtechnologies and microdevices to apply cell compression, in comparison to off-chip bulk systems. Due to their unique features, microfluidic systems developed to apply compressive forces on single cells, in 2D and 3D culture models, and compression in cancer microenvironments are emphasized. Research efforts in this field can help the development of mechanoceuticals in the future.

Capillaric field effect transistors.

Controlling fluid flow in capillaric circuits is a key requirement to increase their uptake for assay applications. Capillary action off-valves provide such functionality by pushing an occluding bubble into the channel using a difference in capillary pressure. Previously, we utilized the binary switching mode of this structure to develop a powerful set of fundamental fluidic valving operations. In this work, we study the transistor-like qualities of the off-valve and provide evidence that these structures are in fact functionally complementary to electronic junction field effect transistors. In view of this, we propose the new term capillaric field effect transistor to describe these types of valves. To support this conclusion, we present a theoretical description, experimental characterization, and practical application of analog flow resistance control. In addition, we demonstrate that the valves can also be reopened. We show modulation of the flow resistance from fully open to pinch-off, determine the flow rate-trigger channel volume relationship and demonstrate that the latter can be modeled using Shockley's equation for electronic transistors. Finally, we provide a first example of how the valves can be opened and closed repeatedly.

Biomechanical responses of encysted zoospores of the oomycete Achlya bisexualis to hyperosmotic stress are consistent with an ability to turgor regulate.

Zoospores are motile, asexual reproductive propagules that enable oomycete pathogens to locate and infect new host tissue. While motile, they have no cell wall and maintain tonicity with their external media using water expulsion vacuoles. Once they locate host tissue, they encyst and form a cell wall, enabling the generation of turgor pressure that will provide the driving force for germination and invasion of the host. It is not currently known how these spores respond to the osmotic stresses that might arise due to different environments on and around their hosts that have different osmotic strengths. We have made microaspiration (MA) measurements on > 800 encysted zoospores and atomic force microscopy (AFM) measurements on 12 encysted zoospores to determine their mechanical properties and how these change after hyperosmotic stress. Two types of encysted zoospores (Type A and Type B) were produced from the oomycete Achlya bisexualis, that differed in their morphology and response. With a small hyperosmotic stress (using 0.1 and 0.2 M sorbitol to give media osmolality changes of 155.4 and 295.6 mOsmol/kg), Type A zoospores initially became stiffer, with an increase in the Young's modulus (E) over 30 mins from 0.16 MPa to 0.25 and 0.22 MPa respectively. E then returned to its original value after 120 min. With a greater osmotic stress (using 0.3, 0.4 and 0.5 M sorbitol to give media osmolality changes of 438.2, 587.2 and 787.6 mOsmol/kg) the reverse occurred, with an initial decrease in E over 30 - 60 mins to values of 0.1, 0.08 and 0.09 MPa respectively, before recovery to the original value after 120 min. In 0.5 M sorbitol this recovery was only observed with AFM, but not with MA. Type B zoospores, which may be primary/secondary spores about to release secondary/tertiary spores, or else spores that were damaged during encystment, initially stiffened in response to the lower hyperosmotic stresses with a slight increase in E (from 0.077 to 0.1 MPa after 15 min (with both 0.1 and 0.2 M sorbitol) before recovering to the original value after 60 min. These spores showed no change in response to the higher osmotic stresses. The responses of the Type A spores are consistent with rapid changes in cell wall thickness and a turgor regulation mechanism. Turgor regulation is further supported by microscopic observations of the Type A spores showing protoplast retraction from the cell wall followed by deplasmolysis, coupled with measurements of spore volume. As far as we are aware this is the first demonstration of turgor regulation, not just in encysted zoospores, but in oomycetes in general.

A dual-flow RootChip enables quantification of bi-directional calcium signaling in primary roots.

One sentence summary: Bi-directional-dual-flow-RootChip to track calcium signatures in Arabidopsis primary roots responding to osmotic stress. Plant growth and survival is fundamentally linked with the ability to detect and respond to abiotic and biotic factors. Cytosolic free calcium (Ca2+) is a key messenger in signal transduction pathways associated with a variety of stresses, including mechanical, osmotic stress and the plants' innate immune system. These stresses trigger an increase in cytosolic Ca2+ and thus initiate a signal transduction cascade, contributing to plant stress adaptation. Here we combine fluorescent G-CaMP3 Arabidopsis thaliana sensor lines to visualise Ca2+ signals in the primary root of 9-day old plants with an optimised dual-flow RootChip (dfRC). The enhanced polydimethylsiloxane (PDMS) bi-directional-dual-flow-RootChip (bi-dfRC) reported here adds two adjacent inlet channels at the base of the observation chamber, allowing independent or asymmetric chemical stimulation at either the root differentiation zone or tip. Observations confirm distinct early spatio-temporal patterns of salinity (sodium chloride, NaCl) and drought (polyethylene glycol, PEG)-induced Ca2+ signals throughout different cell types dependent on the first contact site. Furthermore, we show that the primary signal always dissociates away from initially stimulated cells. The observed early signaling events induced by NaCl and PEG are surprisingly complex and differ from long-term changes in cytosolic Ca2+ reported in roots. Bi-dfRC microfluidic devices will provide a novel approach to challenge plant roots with different conditions simultaneously, while observing bi-directionality of signals. Future applications include combining the bi-dfRC with H2O2 and redox sensor lines to test root systemic signaling responses to biotic and abiotic factors.

Platforms for High-Throughput Screening and Force Measurements on Fungi and Oomycetes.

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

New flow control systems in capillarics: off valves.

Capillary systems are a promising technology for point-of-care microfluidics, since they are pre-programmable and self-powered. This work introduces "off valves" as a key building block for capillaric circuits, providing easy-to-use, multi-purpose valving functionality and autonomous flow control. To this end we present a set of switching valve designs that use trigger channels and liquid input alone to close or open connections between channels in a highly controllable fashion. The key element of all these valve designs is a new off trigger valve, which is characterised in detail here and holds the potential for transistor-like switching and resistance tuning. As an example for the potential applications of switching valves, we demonstrate how they can be used for flow resistance control in a complex microfluidic circuit and for sequential chemical loading into a reaction chamber. Use of the switching valves for the latter in particular allowed for the tuning of incubation times and volumetric measurement, thus confirming applicability of the valves for automated and self-powered immunoassays in point-of-care environments.

Microfluidic platform for integrated compartmentalization of single zoospores, germination and measurement of protrusive force generated by germ tubes.

This paper describes the design, fabrication and characterisation of a novel monolithic lab-on-a-chip (LOC) platform combining the trapping and germination of individual zoospores of the oomycete Achlya bisexualis with elastomeric micropillar-based protrusive force sensing. The oomycetes are of significant interest due to their pathogenic capabilities, which can have profound ecological and economic impacts. Zoospore encystment and germination via a germ tube play a key role in their pathogenicity. Our platform enables the study of these processes at a single cell level through hydrodynamic trapping of zoospores and their individual compartmentalization via normally closed pneumatic membrane microvalves. Valve geometry was optimized and media exchange characterized during dynamic valve operations to enhance the capture-to-growth ratio. We demonstrate germination of A. bisexualis zoospores on the platform and report three distinct germination patterns. Once germinated, germ tubes grew down growth channels towards single elastomeric micropillars. Tracking of pillar movement allowed for the measurement of microNewton range protrusive forces imparted by the tips of the germ tubes. Results indicate that the forces generated by the germ tubes are smaller than those exerted by mature hyphae. Through the use of parallel traps, channels and pillars on the same device, the platform enables high-throughput screening (HTS) of zoospores and their generation of protrusive force, an essential component of their infective capability. Due to its versatility, it will also allow for the screening of naturally bioactive compounds and the development of new biocontrol strategies for oomycetes, and morphologically similar fungal infections, as an alternative to agrochemicals.

Art-on-a-Chip: Preserving Microfluidic Chips for Visualization and Permanent Display.

"After a certain high level of technical skill is achieved, science and art tend to coalesce in aesthetics, plasticity, and form. The greatest scientists are always artists as well." said Albert Einstein. Currently, photographic images bridge the gap between microfluidic/lab-on-a-chip devices and art. However, the microfluidic chip itself should be a form of art. Here, novel vibrant epoxy dyes are presented in combination with a simple process to fill and preserve microfluidic chips, to produce microfluidic art or art-on-a-chip. In addition, this process can be used to produce epoxy dye patterned substrates that preserve the geometry of the microfluidic channels-height within 10% of the mold master. This simple approach for preserving microfluidic chips with vibrant, colorful, and long-lasting epoxy dyes creates microfluidic chips that can easily be visualized and photographed repeatedly, for at least 11 years, and hence enabling researchers to showcase their microfluidic chips to potential graduate students, investors, and collaborators.

Complex Geometry Cellulose Hydrogels Using a Direct Casting Method.

To facilitate functional hydrogel part production using the indirect wax mould method, it is necessary to understand the relationships between materials, process and mould removal. This research investigated the thermophysical properties, wettability and surface roughness of wax template moulds in the production of cellulose hydrogel objects. Cellulose gel was thermally formed and shaped in three different wax moulds-high melting point paraffin, sacrificial investment casting wax and Solidscape® wax-by physical cross-linking of polymer networks of cellulose solution in NaOH/urea aqueous solvent. All three wax moulds were capable of casting cellulose hydrogel objects. Cellulose gelling time was reduced by increasing the temperature. Thus, the mould melting temperature had a direct effect on the gelling time. It was found that mould removal time varied based on the contact angle (CA) of the cellulose solution and the mould, and based on the melting point of the mould. A higher CA of cellulose solution on the wax moulds resulted in faster mould removal. When melting the wax in 90 °C water, high melting point paraffin, sacrificial investment casting and Solidscape® wax took about 3, 2 and 1½ h, respectively, to remove the moulds from the cellulose gel.

Replicating Arabidopsis Model Leaf Surfaces for Phyllosphere Microbiology.

Artificial surfaces are commonly used in place of leaves in phyllosphere microbiology to study microbial behaviour on plant leaf surfaces. These surfaces enable a reductionist approach to be undertaken, to enable individual environmental factors influencing microorganisms to be studied. Commonly used artificial surfaces include nutrient agar, isolated leaf cuticles, and reconstituted leaf waxes. Recently, replica surfaces mimicking the complex topography of leaf surfaces for phyllosphere microbiology studies are appearing in literature. Replica leaf surfaces have been produced in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS). However, none of these protocols are suitable for replicating fragile leaves such as of the model plant Arabidopsis thaliana. This is of importance, as A. thaliana is a model system for molecular plant genetics, molecular plant biology, and microbial ecology. To overcome this limitation, we introduce a versatile replication protocol for replicating fragile leaf surfaces into PDMS. Here we demonstrate the capacity of our replication process using optical microscopy, atomic force microscopy (AFM), and contact angle measurements to compare living and PDMS replica A. thaliana leaf surfaces. To highlight the use of our replica leaf surfaces for phyllosphere microbiology, we visualise bacteria on the replica leaf surfaces in comparison to living leaf surfaces.

Comparison of replica leaf surface materials for phyllosphere microbiology.

Artificial surfaces are routinely used instead of leaves to enable a reductionist approach in phyllosphere microbiology, the study of microorganisms residing on plant leaf surfaces. Commonly used artificial surfaces include, flat surfaces, such as metal and nutrient agar, and microstructured surfaces, such as isolate leaf cuticles or reconstituted leaf waxes. However, interest in replica leaf surfaces as an artificial surface is growing, as replica surfaces provide an improved representation of the complex topography of leaf surfaces. To date, leaf surfaces have predominantly been replicated for their superhydrophobic properties. In contrast, in this paper we investigated the potential of agarose, the elastomer polydimethylsiloxane (PDMS), and gelatin as replica leaf surface materials for phyllosphere microbiology studies. Using a test pattern of pillars, we investigated the ability to replicate microstructures into the materials, as well as the degradation characteristics of the materials in environmental conditions. Pillars produced in PDMS were measured to be within 10% of the mold master and remained stable throughout the degradation experiments. In agarose and gelatin the pillars deviated by more than 10% and degraded considerably within 48 hours in environmental conditions. Furthermore, we investigated the surface energy of the materials, an important property of a leaf surface, which influences resource availability and microorganism attachment. We found that the surface energy and bacterial viability on PDMS was comparable to isolated Citrus × aurantium and Populus × canescens leaf cuticles. Hence indicating that PDMS is the most suitable material for replica leaf surfaces. In summary, our experiments highlight the importance of considering the inherent material properties when selecting a replica leaf surface for phyllosphere microbiology studies. As demonstrated, a PDMS replica leaf offers a control surface that can be used for investigating microbe-microbe and microbe-plant interactions in the phyllosphere, which will enable mitigation strategies against pathogens to be developed.

Towards Point-of-Care Insulin Detection.

Good glucose management through an insulin dose regime based on the metabolism of glucose helps millions of people worldwide manage their diabetes. Since Banting and Best extracted insulin, glucose management has improved due to the introduction of insulin analogues that act from 30 minutes to 28 days, improved insulin dose regimes, and portable glucose meters, with a current focus on alternative sampling sites that are less invasive. However, a piece of the puzzle is still missing-the ability to measure insulin directly in a Point-of-Care device. The ability to measure both glucose and insulin concurrently will enable better glucose control by providing an improved estimate for insulin sensitivity, minimizing variability in control, and maximizing safety from hypoglycaemia. However, direct detection of free insulin has provided a challenge due to the size of the molecule, the low concentration of insulin in blood, and the selectivity against interferants in blood. This review summarizes current insulin detection methods from immunoassays to analytical chemistry, and sensors. We also discuss the challenges and potential of each of the methods towards Point-of-Care insulin detection.

An elastomeric micropillar platform for the study of protrusive forces in hyphal invasion.

Oomycetes and fungi are microorganisms whose pathogenic (invasive) growth can cause diseases that are responsible for significant ecological and economic losses. Such growth requires the generation of a protrusive force, the magnitude and direction of which involves a balance between turgor pressure and localised yielding of the cell wall and the cytoskeleton. To study invasive growth in individual hyphae we have developed a lab-on-a-chip platform with integrated force-sensors based on elastomeric polydimethylsiloxane (PDMS) micro-pillars. With this platform we are able to measure protrusive force (both magnitude and direction) and hyphal morphology. To show the usefulness of the platform, the oomycete Achlya bisexualis was inoculated and grown on a chip. Growth of individual hyphae into a micro-pillar revealed a maximum total force of 10 µN at the hyphal tip. The chips had no discernible effect on hyphal growth rates, but hyphae were slightly thinner in the channels on the chips compared to those on agar plates. When the hyphae contacted the pillars tip extension decreased while tip width increased. A. bisexualis hyphae were observed to reorient their growth direction if they were not able to bend and effectively grow over the pillars. Estimates of the pressure exerted on a pillar were 0.09 MPa, which given earlier measures of turgor of 0.65 MPa would indicate low compliance of the cell wall. The platform is adaptable to numerous cells and organisms that exhibit tip-growth. It provides a useful tool to begin to unravel the molecular mechanisms that underlie the generation of a protrusive force.