Macdara Glynn

Centrifugal microfluidics for cell analysis.

Over the past two decades, centrifugal microfluidic systems have successfully demonstrated their capability for robust, high-performance liquid handling to enable modular, multi-purpose lab-on-a-chip platforms for a wide range of life-science applications. Beyond the handling of homogeneous liquids, the unique, rotationally controlled centrifugal actuation has proven to be specifically advantageous for performing cell and particle handling and assays. In this review we discuss technologies to implement two important steps for cell handling, namely separation and capturing/counting.

Centrifugo-Magnetophoretic Purification of CD4+ Cells from Whole Blood Toward Future HIV/AIDS Point-of-Care Applications

In medical diagnostics, detection of cells exhibiting specific phenotypes constitutes a paramount challenge. Detection technology must ensure efficient isolation of (often rare) targets while eliminating nontarget background cells. Technologies exist for such investigations, but many require high levels of expertise, expense, and multistep protocols. Increasing automation, miniaturization, and availability of such technologies is an aim of microfluidic lab-on-a-chip strategies. To this end, we present an integrated, dual-force cellular separation strategy using centrifugo-magnetophoresis. Whole blood spiked with target cells is incubated with (super-)paramagnetic microparticles that specifically bind phenotypic markers on target cells. Under rotation, all cells sediment into a chamber located opposite a co-rotating magnet. Unbound cells follow the radial vector, but under the additional attraction of the lateral magnetic field, bead-bound target cells are deflected to a designated reservoir. This multiforce separation is continuous and low loss. We demonstrate separation efficiently up to 92% for cells expressing the HIV/AIDS relevant epitope (CD4) from whole blood. Such highly selective separation systems may be deployed for accurate diagnostic cell isolations from biological samples such as blood. Furthermore, this high efficiency is delivered in a cheap and simple device, thus making it an attractive option for future deployment in resource-limited settings.

Paper imbibition for timing of multi-step liquid handling protocols on event-triggered centrifugal microfluidic lab-on-a-disc platforms

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.

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.

Rapid and cost‐efficient enumeration of rare cancer cells from whole blood by low‐loss centrifugo‐magnetophoretic purification under stopped‐flow conditions

We present a substantially improved design and functionality of a centrifugo‐magnetophoretic platform which integrates direct immunoseparation and cost‐efficient, bright‐field detection of cancer cells in whole blood. All liquid handling takes place in a disposable cartridge with geometry akin to a conventional compact disc (CD). The instrumentation required to process such a “lab‐on‐a‐disc” cartridge can be as simple and cost‐efficient as the rotor on a common optical disc drive. In a first step, target cells in a blood sample are specifically bound to paramagnetic microbeads. The sample is then placed into the disc cartridge and spun. In the second step, magnetically tagged target cells are separated by a co‐rotating, essentially lateral magnetic field from the background population of abundant blood cells, and also from unbound magnetic beads. A stream of target cells centrifugally sediments through a stagnant liquid phase into a designated detection chamber. The continuous, multiforce immunoseparation proceeds very gently, i.e. the mechanical and hydrodynamic stress to the target cells is minimized to mitigate the risk of cell loss by collective entrapment in the background cells or vigorous snapping against a wall. We successfully demonstrate the extraction of MCF7 cancer cells at concentrations as low as 1 target cell per μl from a background of whole blood, with capture efficiencies of up to 88%. Its short time‐to‐answer is a notable characteristic of this system, with 10% of target cells collected in the first minute after their loading to the system and the remainder captured within the following 10 min. All the above‐mentioned factors synergetically combine to leverage the development of a prospective point‐of‐care device for CTC detection.

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.

A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion

We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C - MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50 μM. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50 μM. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization.

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.

SIZE- and deformability-based particle sorting by strategic design of obstacle arrays in continuous centrifugal sedimentation mode

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.

A portable optical reader and wall projector towards enumeration of bio-conjugated beads or cells

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.