Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic “Lab-on-a-Disc” cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are ‘low-pass’, i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both ‘hydrostatically’ and ‘hydrodynamically’ triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, ‘dual siphon’ configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.

We introduce a novel instrument controlled valving scheme for centrifugal platforms which is based upon xurography. In a first approach, which is akin to previously presented event-triggered flow control, the valves are composed of a pneumatic chamber sealed by a dissolvable film (DF) and by a pierceable membrane. Liquid is initially prevented from wetting the DF by the counter pressure of a trapped gas. Via a channel, this pocket is pneumatically connected to a vent, sealed by the pierceable membrane, located on the top surface of the disc. By scouring the top surface of the disc, along a pre-defined track by a robotic knife-cutter, the trapped gas is released and so the liquid can wet and disintegrate the DF. In order to automate assay protocols without the need to integrate DFs, we extend this xurography-based flow control concept by selective venting of chambers subjected to pneumatic over-pressure or vacuum suction. Unlike most instrument controlled flow-control mechanisms, in this approach to valve actuation can occur during disc rotation. To demonstrate the potential of this flow control approach, we designed a disc architecture to automate the liquid handling as the backbone of a biplex liver assay panel. We demonstrate valve actuation during rotation, using the robotic arm, using this disc with visualisation via dyed water. We then demonstrate the biplex liver assay, using calibration reagent, by stopping the disc and manually piercing the membrane to actuate the same valves.

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Lab-on-a-Disc (LoaD) has great potential for applications in decentralised bioanalytical testing where speed and robustness are critical. Here, a disc-shaped microfluidic chip is rotated to pump liquid radially outwards; thus, all microfluidic structures must be fitted into the available radial length. To overcome this limitation, several centripetal pumping technologies have been developed. In this work, we combine buoyancy pumping, enabled by displacing aqueous samples and reagents centripetally inwards by a dense liquid (fluorocarbon FC-40), with dissolvable film (DF) to automate a multi-step assay. The DF dissolves in the presence of water but is not in contact with the FC-40. Therefore, the FC-40 can be stored behind the DF membranes and is autonomously released by contact with the arriving aqueous sample. Using this technology, tasks such as blood centrifugation can be located on the disc periphery where ‘disc real estate’ is less valuable and centrifugal forces are higher. To demonstrate this, we use the combination of the buoyancy-driven centripetal pumping with DF barriers to implement a fully automated multi-parameter diagnostic assay on the LoaD platform. The implemented steps include plasma extraction from a structure, automatically triggered metering/aliquoting, and the management of five onboard stored liquid reagents. Critically, we also demonstrate highly accurate aliquoting of reagents using centripetal pumping. We also provide a mathematical model to describe the pumping mechanism and apply lumped-element modelling and Monte Carlo simulation to estimate errors in the aliquoting volumes caused by manufacturing deviations.