Microfluidics in Haemostasis: A Review.

Haemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. As leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of haemostatic disorders. The field of microfluidic and Lab on a Chip (LOC) technologies is rapidly advancing and the important role of miniaturised diagnostics is becoming more evident in the healthcare system, with particular importance in near patient testing (NPT) and point of care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges which have the knock-on effect of improving health care and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of haemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing micro-channels are reviewed in the context of application to the field of haemostasis.

Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs.

We report the development of a microfluidic device for the rapid assay in whole blood of interfacial platelet-protein interactions indicative of the efficacy of antiplatelet drugs, for example, aspirin and Plavix, two of the world's most widely used drugs, in patients with cardiovascular disease (CVD). Because platelet adhesion to surface-confined protein matrices is an interfacial phenomenon modulated by fluid shear rates at the blood/protein interface, and because such binding is a better indicator of platelet function than platelet self-aggregation, we designed, fabricated, and characterized the performance of a family of disposable, self-powered microfluidic chips with well-defined flow and interfacial shear rates suitable for small blood volumes (≤200 µL). This work demonstrates that accurate quantification of cell adhesion to protein matrices, an important interfacial biological phenomenon, can be used as a powerful diagnostic tool in those with CVD, the world's leading cause of death. To enable such measurements, we developed a simple technique to fabricate single-use self-powered chips incorporating shear control (SpearChips). These parallel-plate flow devices integrate on-chip vacuum-driven blood flow, using a predegassed elastomer component to obviate active pumping, with microcontact-printed arrays of 6-µm-diameter fluorescently labeled fibrinogen dots on a cyclic olefin polymer base plate as a means to quantitatively count platelet-protein binding events. The use of SpearChips to assess in whole blood samples the effects of GPIIb/IIIa and P2Y12 inhibitors, two important classes of "antiplatelet" drugs, is reported.

Programmable fluidic networks on centrifugal microfluidic discs.

BACKGROUND: Biomedical diagnostic and lab automation solutions built on the Lab-on-a-Disc (LoaD) platform has great potential due to their independence from specialised micro-pumps and their ease of integration, through direct pipetting, with manual or automated workflows. However, a challenge for all microfluidic chips is their cost of manufacture when each microfluidic disc must be customized for a specific application. In this paper, we present centrifugal discs with programmable fluidic networks. RESULTS: Based on dissolvable film valves, we present two technologies. The first, based on recently introduced pulse-actuated dissolvable film valves, is a centrifugal disc which, depending on how it is loaded, is configured to perform either six sequential reagent releases through one reaction chamber or three sequential reagent releases through two reaction chambers. In the second approach, we use the previously introduced electronic Lab-on-a-Disc (eLoaD) wireless valve array, which can actuate up to 128 centrifugo-pneumatic dissolvable film valves in a pre-defined sequence. In this approach we present a disc which can deliver any one of 8 reagent washes to any one of four reaction chambers. We use identical discs to demonstrate the first four sequential washes through two reaction chambers and then two sequential washes through four reaction chambers. SIGNIFICANCE: These programmable fluidic networks have the potential to allow a single disc architecture to be applied to multiple different assay types and so can offer a lower-cost and more integrated alternative to the standard combination of micro-titre plate and liquid handling robot. Indeed, it may even be possible to conduct multiple different assays concurrently. This can have the effect of reducing manufacturing costs and streamlining supply-chains and so results in a more accessible diagnostic platform.

Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

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.

Development of a fluorescent method for detecting the onset of coagulation in human plasma on microstructured lateral flow platforms.

Microfluidic devices and microsystems have been used to develop blood coagulation monitoring devices for point of care diagnostic use. However, many of them suffer from inherent variability and imprecision, partly due to the fact that they only detect changes in bulk clotting properties and do not reflect the microscopic nature of blood coagulation. This work demonstrates microstructured lateral flow platforms used in combination with fluorescently labelled fibrinogen to detect microscopic clot formation. Plasma samples applied to platforms modified with coagulation activation reagents and fluorescent fibrinogen produced changes in fluorescence intensity due to incorporation of the fluorophore into the forming microclots. It was found that the change in the distribution of the fluorescence within the sample over time was an excellent predictor of the onset of coagulation, which could be used to determine the clotting time. The impact of various assay parameters was optimised and the assay was shown to be capable of measuring the effect of heparin concentration on blood clotting time from 0 to 1.5 U mL(-1).

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.

Microfluidic device to study arterial shear-mediated platelet-surface interactions in whole blood: reduced sample volumes and well-characterised protein surfaces.

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on well-characterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 µL of whole human blood per assay. The device's parallel-plate flow chamber, with 0.1 mm² cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm² cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.

3D Printing of Metallic Microstructured Mould Using Selective Laser Melting for Injection Moulding of Plastic Microfluidic Devices.

A new method, a 3D printing technique, in particular, selective laser melting (SLM), has been used to fabricate moulds for the injection moulding of thermoplastic microfluidic chips that are suitable for prototyping and early stage scale-up. The micro metallic patterns are printed on to a pre-finished substrate to form a microstructured mould. The dimensional accuracy, surface morphology, bonding strength between the printed patterns and substrate, as well as the microstructure of micro features were all characterized. A microfluidic mould was successfully printed and used directly for injection moulding of cyclic olefin copolymer (COC) microfluidic chips, which were used subsequently to successfully monitor nitrite concentrations in environmental water. The characterization indicated that this new process can be used for fast fabrication of mould tools for injection moulding/hot embossing microfluidic devices. It is faster, more flexible and less expensive than conventional micro-machining processes, although the accuracy and finish are still needed to improve though process optimization and hybrid SLM and machining processes.

Precision control of flow rate in microfluidic channels using photoresponsive soft polymer actuators.

A novel approach that allows control of flow in microfluidic channels with unsurpassed performance using light is described. Valve structures have been created using photoresponsive hydrogels based on spiropyran-functionalised pNIPAAm hydrogels photopolymerised around pillar structures within the channels. Valve actuation is controlled from outside the fluidic system using externally located LEDs. Highly precise and accurate flow rates can be selected by passing real-time flow rate measurements into a PID algorithm. The optimised algorithm also minimises overshoot of the selected flow rate, eliminates flow rate drift, and improves the system response time. In addition to the dramatic improvements in flow rate control, the set up enables the polymer actuation behaviour to be rapidly characterised. The power supply to the LED also provides a useful system diagnostic for monitoring the performance of the valve over time. For example, degradation in the valve actuation due to photodegradation will manifest as an increasing power requirement over time, enabling predictive failure thresholds to be established for particular actuator designs and polymer compositions.

Computational Tracking of Shear-Mediated Platelet Interactions with von Willebrand Factor.

The imaging of shear-mediated dynamic platelet behavior interacting with surface-immobilized von Willebrand factor (vWF) has tremendous potential in characterizing changes in platelet function for clinical diagnostics purposes. However, the imaging output, a series of images representing platelets adhering and rolling on the surface, poses unique, non-trivial challenges for software algorithms that reconstruct the positional trajectories of platelets. We report on an algorithm that tracks platelets using the output of such flow run experiments, taking into account common artifacts encountered by previously-published methods, and we derive seven key metrics of platelet dynamics that can be used to characterize platelet function. Extensive testing of our method using simulated platelet flow run data was carried out to validate our tracking method and derived metrics in capturing key platelet-vWF interaction-dynamics properties. Our results show that while the number of platelets present on the imaged area is the leading cause of errors, flow run data from two experiments using whole blood samples showed that our method and metrics can detect platelet property changes/differences that are concordant with the expected biological outcome, such as inhibiting key platelet receptors such as P2Y1, glycoprotein (GP)Ib and GPIIb/IIIa. These findings support the use of our methodologies to characterize platelet function among a wide range of healthy and disease cohorts.

Development of a micro-fluidic manifold for copper monitoring utilising chemiluminescence detection.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).

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 αIIbß (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 device's thrombogenic surface, does not activate platelets, and does not interfere with the action of agonists added to deliberately activate platelets.

Shear-mediated platelet adhesion analysis in less than 100 µl 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.

Robust monolithic silica-based on-chip electro-osmotic micro-pump.

A robust, compact, on-chip, electro-osmotic micro-pump (EOP) for micro-flow analysis, based on parallel, encased, 10 x 0.1 mm I.D. monolithic silica capillary columns has been developed. A 15 x 40 x 2 mm poly(methyl methacrylate) (PMMA) chip, containing a total of nine parallel EOP systems was fabricated, allowing the use of single, double or triple monolithic columns to produce increased flow as required. The monolithic silica was compatible with both aqueous and organic solvents without swelling or shrinking problems, with the triple column EOP capable of generating flow of up to 0.6 microL min(-1) under zero pressure load and over 0.1 microL min(-1) with an applied pressure of ca. 2.4 bar using an applied voltage of just 2 kV. Current generated at the 2 kV applied voltage for a 2 mM acetate buffer solution (pH 4.5) was under 4 microA, allowing stable, bubble-free flow. The developed triple column EOP was incorporated within a micro-fluidic chip (5.0 x 2.0 x 0.4 cm) integrated with a second single 10 x 0.1 mm column EOP, for combined sample injection and simple on-chip micro-flow analysis.

Radioluminescent light source for the development of optical sensor arrays.

A radioluminescent (RL) light source is evaluated for the development of photonically based chemical-responsive sensor arrays (CRSAs). The RL light source is comprised of a strontium-90 (90Sr) radionuclide and a plastic scintillator. The beta particles emitted from the 90Sr generate blue light (lambda(max) = 435 nm) from the plastic scintillator, and the blue light excites the analyte-responsive luminophores within the CRSA. To assess the RL light source utility, we have determined the analytical figures of merit from two tris(4,7'-diphenyl-1,10'-phenathroline)ruthenium(II)-doped xerogel-based sensor platforms: (i) a planar 5 x 5 multielement array and (ii) a discrete sensor element formed on the proximal face of poly(styrene) pillars that have a frustrated cone (frustum) geometry. We compare the performance from each platform when it is excited by a He-Cd laser (442 nm), a blue light-emitting diode (460-470 nm), and the RL light source. The RL light source yields results that are statistically equivalent to results from either electrically powered light source. The RL light source consumes no electrical power, is compact and simple, and has an extremely stable time-averaged signal. The primary trade-offs for these advantages are the RL light source's lower radiant power and the corresponding longer data acquisition times.