Fluigi: an end-to-end software workflow for microfluidic design

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications with implications in health, materials, and sensing. Computational design methodologies allow for increased performance and reliability of these circuits. Major challenges that remain include increasing the scalability and robustness of engineered biological systems and streamlining and automating the synthetic biology workflow of “specify-design-build-test.” I summarize the advances in microfluidic technology, particularly microfluidic large scale integration, that can be used to address the challenges facing each step of the synthetic biology workflow for genetic circuits. Microfluidic technologies allow precise control over the flow of biological content within microscale devices, and thus may provide more reliable and scalable construction of synthetic biological systems. However, adoption of microfluidics for synthetic biology has been slow due to the expert knowledge and equipment needed to fabricate and control devices. I present an end-to-end workflow for a computer-aided-design (CAD) tool, Fluigi, for designing microfluidic devices and for integrating biological Boolean genetic circuits with microfluidics. The workflow starts with a ``netlist" input describing the connectivity of microfluidic device to be designed, and proceeds through placement, routing, and design rule checking in a process analogous to electronic computer aided design (CAD). The output is an image of the device for printing as a mask for photolithography or for computer numerical control (CNC) machining. I also introduced a second workflow to allocate biological circuits to microfluidic devices and to generate the valve control scheme to enable biological computation on the device. I used the CAD workflow to generate 15 designs including gradient generators, rotary pumps, and devices for housing biological circuits. I fabricated two designs, a gradient generator with CNC machining and a device for computing a biological XOR function with multilayer soft lithography, and verified their functions with dye. My efforts here show a first end-to-end demonstration of an extensible and foundational microfluidic CAD tool from design concept to fabricated device. This work provides a platform that when completed will automatically synthesize high level functional and performance specifications into fully realized microfluidic hardware, control software, and synthetic biological wetware

Evolvable Smartphone-Based Point-of-Care Systems For In-Vitro Diagnostics

Recent developments in the life-science -omics disciplines, together with advances in micro and nanoscale technologies offer unprecedented opportunities to tackle some of the major healthcare challenges of our time. Lab-on-Chip technologies coupled with smart-devices in particular, constitute key enablers for the decentralization of many in-vitro medical diagnostics applications to the point-of-care, supporting the advent of a preventive and personalized medicine. Although the technical feasibility and the potential of Lab-on-Chip/smart-device systems is repeatedly demonstrated, direct-to-consumer applications remain scarce. This thesis addresses this limitation. System evolvability is a key enabler to the adoption and long-lasting success of next generation point-of-care systems by favoring the integration of new technologies, streamlining the reengineering efforts for system upgrades and limiting the risk of premature system obsolescence. Among possible implementation strategies, platform-based design stands as a particularly suitable entry point. One necessary condition, is for change-absorbing and change-enabling mechanisms to be incorporated in the platform architecture at initial design-time. Important considerations arise as to where in Lab-on-Chip/smart-device platforms can these mechanisms be integrated, and how to implement them. Our investigation revolves around the silicon-nanowire biological field effect transistor, a promising biosensing technology for the detection of biological analytes at ultra low concentrations. We discuss extensively the sensitivity and instrumentation requirements set by the technology before we present the design and implementation of an evolvable smartphone-based platform capable of interfacing lab-on-chips embedding such sensors. We elaborate on the implementation of various architectural patterns throughout the platform and present how these facilitated the evolution of the system towards one accommodating for electrochemical sensing. Model-based development was undertaken throughout the engineering process. A formal SysML system model fed our evolvability assessment process. We introduce, in particular, a model-based methodology enabling the evaluation of modular scalability: the ability of a system to scale the current value of one of its specification by successively reengineering targeted system modules. The research work presented in this thesis provides a roadmap for the development of evolvable point-of-care systems, including those targeting direct-to-consumer applications. It extends from the early identification of anticipated change, to the assessment of the ability of a system to accommodate for these changes. Our research should thus interest industrials eager not only to disrupt, but also to last in a shifting socio-technical paradigm

A N-ary sorting system using an Integrated Droplet-Digital Microfluidics (ID2M)

Droplet microfluidics has the ability to compartmentalize reactions in sub nano-liter (or pico) volumes that can potentially enable millions of distinct biological assays to be performed on individual cells. Typically, there are two main droplet operations that can be performed with these platforms: cell encapsulation and sorting. Droplet sorting has been a means to select a subset of reactions or activities that can be used for further manipulation and it has been used for single cell analysis and directed evolution. But one of the challenges with these techniques is that these are typically binary sorters – i.e. only relying on two sorting channels. This can limit the range of detecting rare events and to sort based on multiple conditions (i.e. more than two conditions). To alleviate this challenge, we have integrated droplet microfluidics with digital microfluidics to enable n-ary sorting techniques, which we call ‘Integrated Droplet-Digital Microfluidics’ (ID2M). Furthermore, our ‘ID2M’ microfluidic technique also allow on-demand droplet creation and droplet mixing which are two other operations that are difficult to perform in current droplet microfluidic platforms. ID2M is integrated to an automation system for on-demand manipulation of droplets and a spectrometer to detect droplet of interest. The ID2M platform has been validated as a robust on-demand screening system by sorting fluorescein droplets of different concentration with an efficiency of ~ 96 %. The device is further demonstrated for sorting tolerant wild-type and yeast mutants to ionic liquid. We propose that this system has the potential to be used for screening different types of cells and for performing directed evolution on chip

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