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.
Typical Lab-on-a-Disc (LoaD) platforms cannot make a continuous measurement while the disc is spinning; this drawback means that the disc usually must be stopped and aligned with a sensor. This can result in measurement errors in time-dependent assays along with inaccuracies due to liquid displacement and bubble formation in the absence of a stabilising centrifugal field. This paper presents a novel concept for a wirelessly electrified-Lab-on-a-Disc (eLoaD) platform that allows continuous measurement of experimental parameters while the disc is spinning. This platform incorporates all the components needed for measurement within the rotating frame of reference, and bidirectional transmission of data outside this reference frame, thus allowing for online measurement independent of the rotation of the disc. The eLoaD platform is conceived in a modular manner whereby an interchangeable and non-disposable 'Application Disc' can be fitted to the eLoaD platform and so the system can be adapted for a range of optical, electrochemical and other measurement types. As an application example, optical readout, using the Application Disc fitted with a silicon photomultiplier, is demonstrated using a tagged chemiluminescent antibody, which is commonly used, for instance, in ELISA assays. The precision of the eLoaD platform is >94%, while its accuracy, when compared to a commercial benchtop luminometer, is higher than 96%. The modular design of this platform will permit extension of this technology to many other LoaD applications.
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.
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