Development of Microfluidic Instrumentation for Application in the Diagnosis of Rare Anaemias

Globally, the number of children born every year with a rare anaemia exceeds 500,000. The symptoms of rare anaemias range, depending on the mutation, from mild to severe, and in many cases prove to be fatal. The geographical prevalence of rare anaemias is concentrated in developing countries where resources available for diagnosis and treatment are scarce. The gold standard diagnosis of rare anaemia requires a three-tier investigation which is costly and not readily available in the areas most afflicted. As such, there is a need for a low-cost and user friendly method of diagnosis for these diseases. This thesis investigates the diagnostic abilities of a bio-chemical assay that exposes red blood cells to a low pH shock using microfluidic techniques. This involved the development of a novel low-cost microfluidic instrument, which has been named MeCheM, to run Lab-on-a-Chip devices. The experimental techniques and protocols developed are critically reviewed using healthy blood samples as the control. The results from the control population establish baselines for comparison against the diseased samples. Subsequently, the developed methods are investigated for diagnostic capabilities using rare anaemia blood samples. The results from these investigations suggest that there are observable differences for the developed Flow Test in the case of the Thalassaemia and Hereditary Spherocytosis disorders. Similarly, the developed Cell-Surface Adhesion measurements highlighted significant differences among the Sickle Cell samples. Additionally, secondary investigations indicated correlations between the gold standard Red Blood Cell Count and the RBC Count as measured using MeCheM, and Mean Corpuscular Volume and Average Cell Projected Area (pre-acid addition). The development of MeCheM, a novel microfluidic instrument, as a stand-alone device is a key output from this body of work

Reciprocating, buoyancy-driven radial pumping on centrifugal microfluidic platforms

Centrifugal microfluidic systems bear great potential for applications where ruggedness, portability, ease-of-use, and cost efficiency are critical. However, due to the unidirectional nature of the centrifugal pumping force, the number of sequential process steps which can be integrated on these “Lab-on-a-Disc” (LoaD) devices is limited by their finite radial extension. To significantly widen this bottleneck and thus expand the scope of applications that can be ported on these LoaD platforms, various groups have developed a range of centripetal pumping mechanisms. Here, we present two advancements over our previous efforts in this area by combining buoyancy-based pumping with dissolvable film (DF) valves. First, we present a buoyancy-driven, reciprocating flow of a dense liquid initially located an upper reservoir and a sample in a peripheral reservoir. Secondly, we combine buoyancy-driven centripetal pumping with sample discretization and metering to fully integrate and automate a liquid handling protocol towards implementing a multi-parameter bioassay on a disc

Isolation of white blood cells using paper-triggered dissolvable-film valves on a centrifugal platform

The inherent centrifugation capability of the so-called ‘Lab-on-a-Disc’ (LoaD) platforms is widely used for blood processing during sample preparation. Here we introduce a valving technique which ena-bles rotational control of paper wetting to actuate dissolvable film (DF) valves. This mechanism is applied to the separation of whole blood into its chief constituents; plasma, leukocytes and erythrocytes

Laboratory unit operations on centrifugal lab-on-a-disc cartridges using dissolvable-film enabled flow control

The suitability of the centrifugal “lab-on-a-disc” (LoaD) platform for point-of-use / point-of-care deployment, where ruggedness, portability, rapid turn-around times and ease-of-use are key, has resulted in increased interest from the academic community over the last decade. Recently, a new, so-called event-triggered valving paradigm was introduced which circumvented a number of the limitations of commonly used, rotationally actuated valves and complex instrument-controlled valving. In the dissolvable-film based event-triggered approach, it is the liquid movement about a disc which actuates a valve; thus enabling the concatenating of a number of liquid handling operations into an automated cascade. Functioning broadly independent of spin rate, the number of discrete valving operations is only limited by the available disc real-estate. In this work we present this valving paradigm to control a network of discrete Laboratory Unit Operations (LUOs) which, with experienced design, can be integrated together to implement complex fluidic assays. We describe how these valves can be configured, using Boolean-like network relationships, to implement LUOs such as sequential washing steps. We also describe how these valves can be configured to enable metering, mixing and selective routing of liquid flows. Finally, we describe how these valves can be configured to provide accurate temporal control of LUOs; thus providing an entire suite of process control technology which can be used to enable many bio-assays

Rotational-pulse actuated dissolvable-film valves for automated purification of total RNA from E. Coli

In this work we report for the first time on a repertoire of valving technologies which are combined to enable automated purification of total RNA from cell homogenate. Process control is implemented us-ing rotational-pulse actuated dissolvable-film (DF) valves; where the order of valve actuation is deter-mined by the disc architecture while the timing of valve actuation is governed by pulses in the spin rate. Selective liquid routing is enabled by combining a heavy, inert and immiscible liquid plug with a DF. The combination of these technologies enables bead-based extraction of amplifiable RNA, with a yield comparable to gold-standard bench-top protocols

Baking-powder driven centripetal pumping controlled by event-triggering of functional liquids

This paper reports radially inbound pumping by the event-triggered addition of water to on-board stored baking powder in combination with valving by an immiscible, high-specific weight liquid on a centrifugal microfluidic platform. This technology allows making efficient use of precious real estate near the center of rotation by enabling the placement of early sample preparation steps as well as reagent reservoirs at the spacious, high-field region on the perimeter of the disc-shaped rotor. This way the number of process steps and assays that can be integrated on these of this “Lab-on-a-Disc” (LoaD) cartridge can be significantly enhanced while maintaining minimum requirements on the intrinsically simple, spindle-motor based instrumentation

Xurography actuated valving for arbitrary timing of centrifugal flow control in parallelized multi-step bioassays

Here we introduce a new, instrument controlled valving scheme for the centrifugal platform which is based upon dissolvable film (DF) technology. Liquid, restrained at any point upon the disc, is prevented from wetting a DF via a trapped gas pocket. From this pocket a pneumatic channel runs to a sealed vent located on the top surface of the disc. Controlled scouring of this seal by a robotic knifecutter permits venting of the trapped gas, and thus actuation of the valve. To demonstrate the potential of these valves, we present a disc developed towards a biplex liver assay panel

Buoyancy-driven centripetal pumping for nested sample preparation in bioassays

In this paper we present a method of buoyancy-driven centripetal pumping utilizing a heavy, water-immiscible liquid to displace an aqueous sample radially inwards against the prevailing centrifugal force. Using formerly introduced dissolvable-film valving, we demonstrate metering and bidirectional periodical pumping of a single sample in the radial direction. We then show integrated sample discretisation, meter-ing and buoyancy-driven inward pumping in a microfluidic structure designed towards multi-parameter bioassays. This structure permits centrifugation of sample on the periphery of a disc (where the centrifugal field p) and proves a high degree of metering accuracy better than 1.2

Centrifugally automated Solid-Phase Extraction of DNA by immiscible liquid valving and chemically powered centripetal pumping of peripherally stored reagents

This paper presents two flow-control technologies for use on centrifugal Lab-on-a-Disc systems. The first, immiscible liquid valving, selectively blocks microfluidic channels using a high-density liquid fluorocarbon (FC- 40). Used with a specific channel geometry, the FC-40 can permit liquid to enter a chamber but prevents it flowing back along the same path and so acts as “liquid” check-valve. The same liquid can be combined with a water-dissolvable film to provide an extremely robust liquid routing structure. The second technology uses CO2 gas, created by wetting of commodity baking powder by water, to centripetally pump liquid from the periphery of the disc to the centre of the disc. The technologies are combined with valving schemes based on strategically placed, solvent-selective dissolvable films (DFs) to demonstrate repeated pumping of a liquid sample from the edge of the disc to the centre of the disc. The flow-control technologies are then combined to demonstrate fully automated Solid-Phase Extraction (SPE) of DNA with reagent storage on the periphery of the disc. We report an extraction efficiency of 47% measured relative to commercial spin-columns

Digital process control of multi-step assays on centrifugal platforms using high-low-high rotational-pulse triggered valving.

Due to their capability for comprehensive sample-to-answer automation, the interest in centrifugal microfluidic systems has greatly increased in industry and academia over the last quarter century. The main applications of these "Lab-on-a-Disc" (LoaD) platforms are in decentralised bioanalytical point-of-use / point-of-care testing. Due to the unidirectional and omnipresent nature of the centrifugal force, advanced flow control is key to coordinate multi-step / multi-reagent assay formats on the LoaD. Formerly, flow control was often achieved by capillary burst valves which require gradual increments of the spin speed of the system-innate spindle motor. Recent advanced introduced a flow control scheme called 'rotational pulse actuated valves'. In these valves the sequence of valve actuation is determined by the architecture of the disc while actuation is triggered by freely programmable upward spike (i.e. Low-High-Low (LHL)) in the rotational frequency. This paradigm shift from conventional 'analogue' burst valves to 'digital' pulsing significantly increases the number of sequential while also improving the overall robustness of flow control. In this work, we expand on these LHL valves by introducing High-Low-High (HLH) pulse-actuated (PA) valving which are actuated by 'downward' spike in the disc spin-rate. These HLH valves are particularly useful for high spin-rate operations such as centrifugation of blood. We introduce two different HLH architectures and then combine the most promising with LHL valves to implement the time-dependent liquid handling protocol underlying a common liver function test panel