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.

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.

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.

Cellular activation and inflammation leading to endothelial dysfunction is associated with cardiovascular disease (CVD). We investigated whether a single cell label-free multi parameter optical interrogation system can detect endothelial cell and endothelial progenitor cell (EPC) activation in vitro and ex vivo , respectively. Cultured human endothelial cells were exposed to increasing concentrations of tumour necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS) before endothelial activation was validated using fluorescence-activated cell sorting (FACS) analysis of inflammatory marker expression (PECAM-1, E-selectin and ICAM-1). A centrifugal microfluidic system and V-cup array was used to capture individual cells before optical measurement of light scattering, immunocytofluorescence, auto-fluorescence (AF) and cell morphology was determined. In vitro , TNF-α promoted specific changes to the refractive index and cell morphology of individual cells concomitant with enhanced photon activity of fluorescently labelled inflammatory markers and increased auto-fluorescence (AF) intensity at three different wavelengths, an effect blocked by inhibition of downstream signalling with Iκβ. Ex vivo , there was a significant increase in EPC number and AF intensity of individual EPCs from CVD patients concomitant with enhanced PECAM-1 expression when compared to normal controls. This novel label-free ‘lab on a disc’ (LoaD) platform can successfully detect endothelial activation in response to inflammatory stimuli in vitro and ex vivo .

Measurement of the height of a packed column of cells or beads, which can be direclty related to the number of cells or beads present in a chamber, is an important step in a number of diagnostic assays. For example, haematocrit measurements may rapidly identify anemia or polycthemia. Recently, user-friendly and cost-efficient Lab-on-a-Chip devices have been developed towards isolating and counting cell sub-populations for diagnostic purposes. In this work, we present a low-cost optical module for estimating the filling level of packed magnetic beads within a Lab-on-a-Chip device. The module is compatible with a previously introduced, disposable microfluidic chip for rapid determination of CD4+ cell counts. The device is a simple optical microscope module is manufactured by 3D printing. An objective lens directly interrogates the height of packed beads which are efficiently isolated on the finger-actuated chip. Optionally, an inexpensive, battery-powered Light Emitting Diode may project a shadow of the microfluidic chip at approximately 50-fold magnification onto a nearby surface. The reader is calibrated with the filling levels of known concentrations of paramagnetic beads within the finger actuated chip. Results in direct and projector mode are compared to measurements from a conventional, inverted white-light microscope. All three read-out methods indicate a maximum variation of 6.5% between methods.

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 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.

From the beginning of the twenty-first century, there has been a shift towards studying biological processes using a holistic rather than a reductionist scientific paradigm thus establishing the approach now named "systems biology" or "systomics". This method of biological investigation represents a synergy where life sciences, systems engineering, and information technology examine the interactions between biological pathways, rather than solely focusing on individual pathways in an isolated manner. To date, systems biology has often studied population averages rather than individual characteristics of cells which might display a significant spread. However, as a single cell is the smallest operational biological unit that encompasses all metabolites necessary for maintaining a viable living entity, the application of systems biology approaches to the study of distinct cells is fast becoming a goal of many research groups. In this chapter we will describe some of the technologies that enable the isolation of individual cells in a form that accommodates systomics studies, the biological methods that are then deployed on such isolated cells to generate system-level information, and finally describe some of the bioinformatics that is specifically directed towards single-cell studies.

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.

In addition detecting circulating tumour cells (CTCs) in blood, the presence of multi-cellular clusters has recently been identified to carry further information pertaining to patient outcome. We present a label-free method of measuring the range and load of clusters in a blood sample using a size-exclusion rail operated by centrifugal microfluidics. A negative selection strategy first enriches clusters from a whole blood sample; these clusters are then processed along the rail where they resolve to a series of collection bins according to size. Analysis of the occupancy of these bins then provides metrics on the cancer-cell load carried in the blood.

In this work we present a centrifugal microfluidic system enabling highly efficient collective trapping and alignment of particles such as microbeads and cells, their multi-colour fluorescent detection and subsequent manipulation by optical tweezers. We demonstrate array-based capture and imaging followed by "cherry-picking" of individual particles, first for fluorescently labelled polystyrene (PS) beads and then for cells. Different cell lines are discriminated based on intracellular as well as surface-based markers.

This paper describes a novel, smart-grid technique for isolating, sorting and capturing cancer cells based on size and deformability by lateral displacement. In stopped-flow, centrifugal sedimentation mode, the bioparticles are sizeselectively displaced along the dynamically spaced grid of the microstructures in a deterministic fashion. Following displacement, particles are captured in claw-like structures for onward processing. This displacement method was first demonstrated for sorting seven bead populations with diameters between 5 μm and 30 μm from a mixture. Then HeLa and melanoma cells spiked in buffer and whole blood were isolated and captured. Finally, we succeeded to sort normal and fixed HeLa cells according to their deformability.

Rotational microfluidic platforms have attracted swiftly growing interest over the last decade due to their suitability for integration and automation of sample preparation and detection. Valving is of pivotal importance on these compact "Lab-on-a-Disc" (LoaD) platforms as all liquids are exposed to the same centrifugal field. A number of valving technologies have been developed to coordinate timing of serial and/or parallel multi-step/multi-liquid assay protocols comprising of laboratory unit operations (LUOs) such as the release, metering and mixing of sample and reagents. So far these valving techniques could be broadly categorised into rotationally controlled or externally actuated schemes. Only recently a new, "event-triggered" flow control has been introduced. In this approach, a valve is opened upon arrival of a liquid at a defined destination on the disc; this innovative mechanism for the first time permits the cascading of LUOs independent of the spin rate. In one technology, dissolvable films (DFs) are configured with a pneumatic chamber to offer function akin to an electrical relay. Dissolving one DF, termed the control film (CF), results in the release of liquid at a distal location through a so-called load film (LF). In this paper, a new method for temporal control of actuating DF-based, event-triggered CFs which are serially aligned at defined distances along a paper strip is introduced. Liquids are transported through the paper strip at a given velocity, thus setting well-defined intervals between subsequent LUOs, e.g. incubation steps. As a proof-of-concept, we present a disc with integrated metering and mixing which can perform a prototypical, 4-fold serial dilution; a common function in bioanalytical protocols. Imbibition of the paper strip sequentially opens five valves for serial dilution and mixing. To illustrate an unprecedented level of on-disc automation, this is followed by a branched cascade of 17 event-triggered valves (for a total of 22 liquid handling steps) which completes the serial dilution protocol.

Cancer is a leading cause of death worldwide, with metastasis responsible for the majority of cancer-related deaths. Circulating tumour cells (CTCs) play a central role in metastasis. Fluorescent silica particles (NPs), of diameter similar to 50 nm which contain a large concentration of Cy5 dye molecules and are extremely bright, have been developed to detect these rare CTCs. Due to this brightness, the particles have superior performance compared to single Cy5 dye molecule labels, for detecting cancer cells. Fluorescence measurements show that the NPs are almost 100 times brighter than the free dye. They do not photobleach as readily and, due to the biocompatible silica surface, they can be chemically modified, layer-by-layer, in order to bind to cells. The choice of these chemical layers, in particular the NP to antibody linker, along with the incubation period and type of media used in the incubation, has a strong influence on the specific binding abilities of the NPs. In this work, NPs have been shown to selectively bind to the MCF-7 cell line by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane by conjugating anti-EpCAM antibody to the NP surface. Results have shown a high signal to noise ratio for this cell line in comparison to a HeLa control line. NP attachment to cells was verified qualitatively with the use of fluorescence microscopy and quantitatively using image analysis methods. Once the system has been optimised, other dyes will be doped into the silica NPs and their use in multiplexing will be investigated.

There is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall prognosis. We present a novel two-stage, stopped-flow, continuous centrifugal sedimentation strategy to measure the size distributions of events (defined here as cells or clusters thereof) in a blood sample. After off-chip removal of red blood cells, healthy white blood cells are sequestered by negative-immunocapture. The purified events are then resolved along a radially inclined rail featuring a series of gaps with increasing width, each connected to a designated outer collection bin. The isolation of candidate events independent of target-specific epitopes is successfully demonstrated for HL60 (EpCAM positive) and sk-mel28 (EpCAM negative) cells using identical protocols and reagents. The propensity to form clusters was quantified for a number of cell lines, showing a negligible, moderate or elevated tendency towards cluster formation. We show that the occupancy distribution of the collection bins closely correlates with the range of cluster sizes intrinsic to the specific cell line.

We report a novel, integrated opto-microfluidic technique for real-time measurements of the individual response of cells in a population to various stimuli. We use this method to study macrophages which are key cellular players in atherosclerosis. Naïve non-activated macrophages (M0) can differentiate into M1 or M2 phenotypic subtypes. In atherosclerosis, M2-macrophages activity is anti-inflammatory while M1-macrophages activity is pro-atherogenic. These cell-type specific changes are measured via their unique Photonic Fingerprints (“PINs”) comprising of scattering, absorbance and auto-fluorescence signals, obtained in a label-free fashion.

We present a disc-based module for rotationally controlled solid-phase purification of RNA from cell lysate. To this end, multi-stage routing of a sequence of aqueous and organic liquids into designated waste and elution reservoirs is implemented by a network of strategically placed, solvent-selective composite valves . Using a bead-based stationary phase at the entrance of the router, we show that total RNA is purified with high integrity from cultured MCF7 and T47D cell lines, human leucocytes and Haemophilus influenzae cell lysates. Furthermore, we demonstrate the broad applicability of the device through the in vitro amplification of RNA purified on-disc using RT-PCR and NASBA. Our novel router will be at the pivot of a forthcoming, fully integrated and automated sample preparation system for RNA-based analysis .