Niamh Kilcawley

Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs.

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.

Baking Powder Actuated Centrifugo-Pneumatic Valving for Automation of Multi-Step Bioassays

We report a new flow control method for centrifugal microfluidic systems; CO₂ is released from on-board stored baking powder upon contact with an ancillary liquid. The elevated pressure generated drives the sample into a dead-end pneumatic chamber sealed by a dissolvable film (DF). This liquid incursion wets and dissolves the DF, thus opening the valve. The activation pressure of the DF valve can be tuned by the geometry of the channel upstream of the DF membrane. Through pneumatic coupling with properly dimensioned disc architecture, we established serial cascading of valves, even at a constant spin rate. Similarly, we demonstrate sequential actuation of valves by dividing the disc into a number of distinct pneumatic chambers (separated by DF membranes). Opening these DFs, typically through arrival of a liquid to that location on a disc, permits pressurization of these chambers. This barrier-based scheme provides robust and strictly ordered valve actuation, which is demonstrated by the automation of a multi-step/multi-reagent DNA-based hybridization assay.

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.

Label-free, spatially multiplexed SPR detection of immunoassays on a highly integrated centrifugal Lab-on-a-Disc platform

As a direct, label-free method, Surface Plasmon Resonance (SPR) detection significantly reduces the needs for liquid handling and reagent storage compared to common enzyme-linked immunosorbent assays (ELISAs), thus enabling comprehensive multiplexing of bioassays on microfluidic sample-to-answer systems. This paper describes a highly integrated centrifugal Lab-on-a-Disc (LoaD) platform for automating the full process chain extending between plasma extraction and subsequent aliquoting to five parallelized reaction channels for quantitative SPR detection by an inexpensive smartphone camera. The entire, multi-step / multi-reagent operation completes within less than 1 h. While the emphasis of this work is on the fluidic automation and parallelization by previously introduced, very robust event-triggered valving and buoyancy-driven centripetal pumping schemes, we successfully implement an immunoglobulin G (IgG) assay; by specific functionalization of the detection surfaces, the same disc layout can readily be customised for immunoassays panels from whole blood.