Simplified fabrication of complex multilayer microfluidics: enabling sophisticated lab-on-a-chip and point-of-care platforms

Complex multilayer microfluidics have generated a lot of interest in recent years. Early research introduced elastomer microvalves and postulated they would bring about a revolution for microfluidic systems, similar in scale to introduction of the transistor for electronic systems. In the following years, many researchers have been active in the use of complex multilayer microfluidic systems, with numerous high impact research outcomes using these systems as precise and active control components, providing fluidic isolation, switching or fluidic actuation, and allowing unprecedented sophistication and precise control and automation of experimental conditions. While application of complex multilayer microfluidic platforms has been demonstrated in numerous research settings, there is little evidence that the technology has become ubiquitously accepted, with a lack of evidence for point-of-care application, or widespread acceptance within the research community. While the advantages that the technology offers have been well documented, the field seems to have failed to gain traction, or facilitate the revolution that was predicted on its introduction. There are various possible explanations for this lack of acceptance, as with any technology, there are caveats to the application of complex multilayer microfluidic systems, however given the broad range of demonstrated applications, it is unlikely that the bottleneck in their application is related to a fundamental application related limitation. In contrast, fabrication technology utilised in realisation of complex multilayer microfluidic systems, has not advanced at the same rate to the multitude of application-based publications in the past decade. This thesis explores the hypothesis that one of the fundamental limiting factors in widespread application of complex multilayer microfluidic systems, is related to the challenges associated with fabrication of these systems. To explore this hypothesis, firstly, a new fabrication approach is introduced which aims to eliminate many of the challenges associated with traditional multilayer fabrication methods, this technique is demonstrated in a proof of concept capacity, fabricating common multilayer microfluidic structures and doing so with surprising ease. Having developed method with simpler fabrication, it is possible to explore whether overcoming the multilayer fabrication bottleneck would allow the advantages inherent to complex multilayer microfluidic systems to be applied to fields which would otherwise be considered prohibitively difficult, if reliant on traditional fabrication methods. This hypothesis is investigated through harnessing the new, simplified fabrication technique to advance point-of-care photonic biosensor research through short term collaborative engagements.  It is found that the use of modular building blocks and the simple, rapid fabrication enables sophisticated microfluidic chip prototypes to be developed in a very short period of time achieving multiple iterations over a matter of weeks and even facilitating collaboration on these integrated platforms remotely. The outcomes of these short-term collaborations have produced publications automating the fluid handling of highly sensitive interferometric waveguide biosensors and environmental control for a single cell analysis platform utilising integrated plasmonic biosensors.       Having demonstrated that simplifying complex microfluidic fabrication can accelerate the development and deployment of these systems to enhance research platforms, the next step was to explore whether this simplified system could also lower the barrier to deployment in a clinical setting. The ability for the fluidic system to handle whole blood was chosen as a deliberately challenging target with great sensitivity to fluid dynamics and large variability in patient samples and environmental factors, requiring large number of replicate devices to determine statistical significance. Here the fabrication technique is applied to enable a study investigating the hemocompatibility of common multilayer control components, paving the way for point of care blood handling devices.  It is shown that not only can the technique be used to rapidly develop platforms that can be used with blood, but the same technique can produce even hundreds of replicates required for limited clinical trials, leading the collaborating clinicians to seriously consider these complex microfluidics for future point of care diagnostics. In Summary, it has been demonstrated that access to complex multilayer microfluidic systems without the fabrication overheads generally associated with these systems can allow their application to areas that would otherwise be prohibitively difficult. The fabrication method presented can allow rapid development, and rapid and reliable deployment to various research applications, while allowing the consistency and throughput required enabling large volume fabrication required for clinical investigations.  The fact that such a large advancement toward real world application within the scope of a single PhD is possible, supports the hypothesis that lowering the barrier to fabricating complex microfluidic devices has the potential to significantly increase their scope of application

An automated optofluidic biosensor platform combining interferometric sensors and injection moulded microfluidics

A primary limitation preventing practical implementation of photonic biosensors within point-of-care platforms is their integration with fluidic automation subsystems. For most diagnostic applications, photonic biosensors require complex fluid handling protocols; this is especially prominent in the case of competitive immunoassays, commonly used for detection of low-concentration, low-molecular weight biomarkers. For this reason, complex automated microfluidic systems are needed to realise the full point-of-care potential of photonic biosensors. To fulfil this requirement, we propose an on-chip valve-based microfluidic automation module, capable of automating such complex fluid handling. This module is realised through application of a PDMS injection moulding fabrication technique, recently described in our previous work, which enables practical fabrication of normally closed pneumatically actuated elastomeric valves. In this work, these valves are configured to achieve multiplexed reagent addressing for an on-chip diaphragm pump, providing the sample and reagent processing capabilities required for automation of cyclic competitive immunoassays. Application of this technique simplifies fabrication and introduces the potential for mass production, bringing point-of-care integration of complex automated microfluidics into the realm of practicality. This module is integrated with a highly sensitive, label-free bimodal waveguide photonic biosensor, and is demonstrated in the context of a proof-of-concept biosensing assay, detecting the low-molecular weight antibiotic tetracycline

Microfluidic platform for separation and extraction of plasma from whole blood using dielectrophoresis

Microfluidic based blood plasma extraction is a fundamental necessity that will facilitate many future lab-on-a-chip based point-of-care diagnostic systems. However, current approaches for providing this analyte are hampered by the requirement to provide external pumping or dilution of blood, which result in low effective yield, lower concentration of target constituents, and complicated functionality. This paper presents a capillary-driven, dielectrophoresis-enabled microfluidic system capable of separating and extracting cell-free plasma from small amounts of whole human blood. This process takes place directly on-chip, and without the requirement of dilution, thus eliminating the prerequisite of preprocessed blood samples and external liquid handling systems. The microfluidic chip takes advantage of a capillary pump for driving whole blood through the main channel and a cross flow filtration system for extracting plasma from whole blood. This filter is actively unblocked through negative dielectrophoresis forces, dramatically enhancing the volume of extracted plasma. Experiments using whole human blood yield volumes of around 180 nl of cell-free, undiluted plasma. We believe that implementation of various integrated biosensing techniques into this plasma extraction system could enable multiplexed detection of various biomarkers

Porous PDMS structures for the storage and release of aqueous solutions into fluidic environments

Typical microfluidic systems take advantage of multiple storage reservoirs, pumps and valves for the storage, driving and release of buffers and other reagents. However, the fabrication, integration, and operation of such components can be difficult. In particular, the reliance of such components on external off-chip equipment limits their utility for creating self-sufficient, stand-alone microfluidic systems. Here, we demonstrate a porous sponge made of polydimethylsiloxane (PDMS), which is fabricated by templating microscale water droplets using a T-junction microfluidic structure. High-resolution microscopy reveals that this sponge contains a network of pores, interconnected by small holes. This unique structure enables the sponge to passively release stored solutions very slowly. Proof-of-concept experiments demonstrate that the sponge can be used for the passive release of stored solutions into narrow channels and circular well plates, with the latter used for inducing intracellular calcium signalling of immobilised endothelial cells. The release rate of stored solutions can be controlled by varying the size of interconnecting holes, which can be easily achieved by changing the flow rate of the water injected into the T-junction. We also demonstrate the active release of stored liquids into a fluidic channel upon the manual compression of the sponge. The developed PDMS sponge can be easily integrated into complex micro/macro fluidic systems and prepared with a wide array of reagents, representing a new building block for self-sufficient microfluidic systems

Active micropump-mixer for rapid antiplatelet drug screening in whole blood

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies

Dynamic drag force based on iterative density mapping: A new numerical tool for three-dimensional analysis of particle trajectories in a dielectrophoretic system

Dielectrophoresis is a widely used means of manipulating suspended particles within microfluidic systems. In order to efficiently design such systems for a desired application, various numerical methods exist that enable particle trajectory plotting in two or three dimensions based on the interplay of hydrodynamic and dielectrophoretic forces. While various models are described in the literature, few are capable of modeling interactions between particles as well as their surrounding environment as these interactions are complex, multifaceted, and computationally expensive to the point of being prohibitive when considering a large number of particles. In this paper, we present a numerical model designed to enable spatial analysis of the physical effects exerted upon particles within microfluidic systems employing dielectrophoresis. The model presents a means of approximating the effects of the presence of large numbers of particles through dynamically adjusting hydrodynamic drag force based on particle density, thereby introducing a measure of emulated particle-particle and particle-liquid interactions. This model is referred to as "dynamic drag force based on iterative density mapping." The resultant numerical model is used to simulate and predict particle trajectory and velocity profiles within a microfluidic system incorporating curved dielectrophoretic microelectrodes. The simulated data are compared favorably with experimental data gathered using microparticle image velocimetry, and is contrasted against simulated data generated using traditional "effective moment Stokes-drag method," showing more accurate particle velocity profiles for areas of high particle density

Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion

Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research

Elastomeric microvalve geometry affects haemocompatibility

This paper reports on the parameters that determine the haemocompatibility of elastomeric microvalves for blood handling in microfluidic systems. Using a comprehensive investigation of blood function, we describe a hierarchy of haemocompatibility as a function of microvalve geometry and identify a "normally-closed" v-gate pneumatic microvalve design that minimally affects blood plasma fibrinogen and von Willebrand factor composition, minimises effects on erythrocyte structure and function, and limits effects on platelet activation and aggregation, while facilitating rapid switching control for blood sample delivery. We propose that the haemodynamic profile of valve gate geometries is a significant determinant of platelet-dependent biofouling and haemocompatibility. Overall our findings suggest that modification of microvalve gate geometry and consequently haemodynamic profile can improve haemocompatibility, while minimising the requirement for chemical or protein modification of microfluidic surfaces. This biological insight and approach may be harnessed to inform future haemocompatible microfluidic valve and component design, and is an advance towards lab-on-chip automation for blood based diagnostic systems