Nigel Kent

Integrated system investigating shear-mediated platelet interactions with von Willebrand factor using microliters of whole blood.

We report an integrated platelet translocation analysis system that measures complex dynamic platelet-protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics. Whole blood flow data from healthy donors at an arterial shear rate (1500 s(-1)) show mean platelet velocities from 8.9+/-1.0 to 12+/-4 microm s(-1). Platelets in blood treated with the antiplatelet agent c7E-Fab fragment spend more than twice as much time in motion as platelets from untreated control blood; the bond dissociation rate constant (k(off)) increases 1.3-fold, whereas mean translocation velocities do not differ. Blood from healthy unmedicated donors was used to assess flow assay reproducibility, donor variability, and the effects of antiplatelet treatment. This integrated system enables reliable, rapid populational quantification of platelet translocation under pathophysiological vascular fluid shear using as little as 150 microl of blood.

Shear-Mediated Platelet Adhesion Analysis in Less Than 100 μ L of Blood: Toward a POC Platelet Diagnostic

We report a microfluidic chip-based hydrodynamic focusing approach that minimizes sample volume for the analysis of cell-surface interactions under controlled fluid-shear conditions. Assays of statistically meaningful numbers of translocating platelets interacting with immobilized von Willebrand factor at arterial shear rates (~1500 s-1) are demonstrated. By controlling spatial disposition and relative flow rates of two contacting fluid streams, e.g., sample (blood) and aqueous buffer, on-chip hydrodynamic focusing guides the cell-containing stream across the protein surface as a thin fluid layer, consuming ~50 μL of undiluted whole blood for a 2-min platelet assay. Control of wall shear stress is independent of sample consumption for a given flow time. The device design implements a mass-manufacturable fabrication approach. Fluorescent labeling of cells enables readout using standard microscopy tools. Customized image-analysis software rapidly quantifies cellular surface coverage and aggregate size distributions as a function of time during blood-flow analyses, facilitating assessment of drug treatment efficacy or diagnosis of disease state.

Fluorescence-Based Blood Coagulation Assay Device for Measuring Activated Partial Thromboplastin Time

The measurement of blood clotting time is important in a range of clinical applications such as assessing coagulation disorders and controlling the effect of various anticoagulant drug therapies. Clotting time tests essentially measure the onset of clot formation which results from the formation of fibrin fibers in the blood sample. However, such assays are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. This work describes a clotting time measurement assay which uses a fluorescent probe to very precisely detect the onset of fibrin clot formation. It uses a microstructured surface which enhances the formation of multiple localized clot loci and which results in the abrupt redistribution of the fluorescent label at the onset of clot formation in both whole blood and plasma. This methodology was applied to the development of an activated partial thromboplastin time (aPTT) test in a lateral flow microfluidic platform and used to monitor the effect of heparin dosage where it showed linearity from 0 to 2 U/mL in spiked plasma samples (R(2)=0.996, n = 3), correlation against gold standard coagulometry of 0.9986, and correlation against standard hospital aPTT in 32 patient samples of 0.78.

Effective Hydrodynamic Shaping of Sample Streams in a Microfluidic Parallel-Plate Flow-Assay Device: Matching Whole Blood Dynamic Viscosity

We report the development of an aqueous buffer system tailored to the fluidic and hemodynamic requirements of our recently reported microfluidic platelet dynamic assay device, which uses hydrodynamic focusing to “shape” a blood sample into a thin flowing layer adjacent to its protein-functionalized surface. By matching the dynamic viscosity of whole blood (3.13 ± 0.08 mPa·s, from healthy donors), the selected buffer minimizes interfacial fluid mixing and better controls shear rate within the device, permitting platelet/protein-surface interaction assays with as little as 50 μL of whole blood. Buffers containing the viscosity-enhancing components bovine serum albumin (BSA), gelofusine/glycine, or histopaque (Ficoll gradient solution) were found not to activate platelets when incubated with blood at concentrations up to 50%, as assessed by flow cytometry quantitation of P-selectin expression and αIIbβ 3 activation. In contrast, glycerol-based buffer activated platelets (two-fold increase in P-selectin levels) at concentrations as low as 10% by volume. BSA- and gelofusine/glycine-based buffers were problematic in preparation and use, and therefore, were not used beyond initial characterization. The histopaque solution selected as the best choice for flow studies stabilizes sample contact with the devices thrombogenic surface, does not activate platelets, and does not interfere with the action of agonists added to deliberately activate platelets.

Development of a low cost microfluidic sensor for the direct determination of nitrate using chromotropic acid in natural waters

Progress towards the development of a miniaturised microfluidic instrument for the direct measurement of nitrate in natural waters and wastewater using chromotropic acid is presented. For the first time, the chromotropic method for nitrate analysis has been transferred to a microfluidic chip configuration that can withstand the extremely acidic nature of the reagent within a field deployable platform. This simple method employs one reagent mixed in a 1 : 1 ratio with the sample to produce a yellow colour absorbing strongly at 430 nm. A stopped flow approach is used which, together with the very rapid kinetics and simple reagent stream, enables an uncomplicated microfluidic design and field deployable platform with a sample throughput of 9 samples per h, limits of detection of 0.70 mg L−1 NO3− and 0.31 mg L−1 NO3− for seawater samples, with a dynamic linear range from 0–80 mg L−1 NO3− and long-term reagent stability of up to 6 months. Validation was achieved by analysing split water samples by the analyser and ion chromatography, resulting in an excellent correlation co-efficient of 0.9969. The fully integrated sensing platform consists of a sample inlet with filter, storage units for chromotropic reagent and standards for self-calibration, pumping system which controls the transport and mixing of the sample, a microfluidic mixing and detection chip, and waste storage, all contained within a ruggedized, waterproof housing. The optical detection system consists of a LED light source with a photodiode detector, which enables sensitive detection of the coloured complex formed. The low cost of the platform coupled with integrated wireless communication makes it an ideal platform for in situ environmental monitoring.

Characterisation of novel refractometric sensing systems

Simulations and experimental results for novel refractometric-sensing platforms are presented here. The first platform is based on a multi-mode interference coupler (MMIC) in which the top and sidewalls of the coupler are exposed to a humidity-sensing enrichment layer. Sensor operation is based on the creation of self-images of the input field into the coupler, at regular intervals along the coupler. This phenomenon is due to interference between the optical modes in MMICs. Changes in the refractive index of the sensing layer cause predictable shifts in the position of the output image, which in turn affects the amount of light coupled into the output waveguide. Sensitivity enhancement has been demonstrated by fabricating longer MMICs capturing higher-ranking self-images, which are shifted more than the first self-image. Consequently, more significant changes in the amount of light coupled to the output are observed for a given refractive index change. The second platform demonstrated is a Multi-Channel Directional Coupler sensor (MCDC). It differs from the MMIC in that the sensing region now consists of multiple single-mode waveguides, which are in close enough proximity to allow light to transfer between the waveguides. Sensitivity dependance on platform length has been investigated and compared with that of the MMIC. The devices have been fabricated by the direct laser writing process on UV curable hybrid sol-gel materials. Such materials allow implementation of planar technology enabling integration on a silicon substrate. Future applications of these platforms include chemical and bio-chemical sensing is the areas of process, environmental and bio-diagnostic monitoring.

Novel polymer platform for enhanced biochip performance

We report the development of enhanced optical platforms for fluorescence-based biosensors. A previous analysis by us has shown that the emission of fluorescence in such a system is highly anisotropic and is preferentially emitted into the substrate over a well-defined angular range, with the result that the light is guided along the substrate via total internal reflection. However, conventional optical biosensors based on fluorescence detection typically employ a detector that is positioned either directly above or directly below the biochip. As a consequence, only a small fraction of the total emitted fluorescence is detected, which impacts adversely on sensor performance. The enhanced biosensor presented here is based on a novel, generic platform specifically designed to overcome the inherent limitations of planar substrates. The platform incorporates custom-designed optical elements, the purpose of which is to redirect the emitted fluorescence onto a detector positioned beneath the biochip. Platforms were fabricated using the polymer processing technique of microinjection moulding. In this paper we demonstrate the ability of this optical system to achieve a 80-fold luminescence capture enhancement. We also demonstrate its effectiveness as an enhanced biosensor platform by carrying out a proof of principle BSA/antiBSA competitive assay. This work has significant implications for the development of mass-producible, highly efficient optical biosensors.

Mitigation and control of the overcuring effect in mask projection micro-stereolithography

Mask Projection micro-Stereolithography (MPμSL) is an additive manufacturing technique capable of producing solid parts with micron-scale resolution from a vat of photocurable liquid polymer resin. Although the physical mechanism remains the same, the process differs from traditional laser-galvanometer based stereolithography (SL) in its use of a dynamic mask UV projector, or digital light processor (DLP), which cures each location within each 3D layer at the same time. One area where MPµSL has garnered considerable attention is in the field of microfluidics and Lab-on-a-Chip, where complex multistep microfabrication techniques adopted from the semiconductor industry are still widely used, and where MPµSL offers the ability to fabricate completely encapsulated fluidic channels in a single step and at low cost [1–3]. However, a significant obstacle exists in the prevention of channel blockage due to overcuring of the polymer resin [4, 5]. Overcuring can be attributed to the so-called ‘back side effect’ [2] which occurs during the build process as light from successive layers penetrates into the resin to a depth greater than the layer thickness. This effect is most prevalent in channels or features oriented horizontally (in a parallel plane to that of the build platform). Currently there are two main approaches in controlling the cure depth; 1. the chemical approach, which involves doping the resin material with a chemical light absorber [6–8]; and 2. by improving the systems hardware and optical elements to improve the homogeneity of the light dosage and control the cure depth [9]. Here we investigate a third approach through modification of the 3D CAD file prior to printing to mitigate for UV light leakage from successive build layers. Although used here in conjunction with the MPμSL technique, this approach can be applied to a range of SL techniques to improve printer resolution and enable production of internal features with higher dimensional accuracy.

Precise Flow-Control Using Photo-Actuated Hydrogel Valves and Pid-controlled LED Actuation

Herein we demonstrate remarkable control of flow within fluidic channels using photo-actuated hydrogel valves. By polymerizing the valves in situ it has been possible to create highly- reproducible valves. Through the use of an LED platform and a PID algorithm we have generated extremely accurate flow control and created prototype devices to document their potential application within the microfluidics field.

Physical integrity of 3D printed parts for use as embossing tools

Abstract On inception, 3D printed parts were typically used at prototyping stage to give the end user/customer a real world concept of how the part may appear when traditional manufacturing techniques were employed for final part fabrication. In this context, mechanical properties such as load bearing capacity or wear rate were not typically of primary concern. This paper investigates, given the advances in 3D printing technology, the potential for using 3D printed parts for high throughput embossing tools. The key mechanical properties for embossing tools are compression and wear rate. To this end, commercially available engineering grade photopolymer materials were characterised in terms of compression and wear using ASTM D695 and ASTM G99 standards respectively. Parts were fabricated via the Polyjet ink-jetting 3D printing technique using the commercially available Connex 260 from Stratasys. Given the nature of the fabrication technique, differences in compressive strength of the material based on orientation of build were also investigated.