Publications by authors named "Jens Ducree"

51 Publications

Siphon-Controlled Automation on a Lab-on-a-Disc Using Event-Triggered Dissolvable Film Valves.

Biosensors (Basel) 2021 Mar 6;11(3). Epub 2021 Mar 6.

School of Physical Sciences, Dublin City University, Dublin 9, Ireland.

Within microfluidic technologies, the centrifugal microfluidic "Lab-on-a-Disc" (LoaD) platform offers great potential for use at the PoC and in low-resource settings due to its robustness and the ability to port and miniaturize 'wet bench' laboratory protocols. We present the combination of 'event-triggered dissolvable film valves' with a centrifugo-pneumatic siphon structure to enable control and timing, through changes in disc spin-speed, of the release and incubations of eight samples/reagents/wash buffers. Based on these microfluidic techniques, we integrated and automated a chemiluminescent immunoassay for detection of the CVD risk factor marker C-reactive protein displaying a limit of detection (LOD) of 44.87 ng mL and limit of quantitation (LoQ) of 135.87 ng mL.
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http://dx.doi.org/10.3390/bios11030073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000095PMC
March 2021

Disease-Relevant Single Cell Photonic Signatures Identify S100β Stem Cells and their Myogenic Progeny in Vascular Lesions.

Stem Cell Rev Rep 2021 Mar 17. Epub 2021 Mar 17.

Vascular Biology and Therapeutics Laboratory, School of Biotechnology Faculty of Science and Health, Dublin City University, Dublin, 9, Ireland.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100β mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- β1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100β vSCs and identified the presence of S100βvSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.
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http://dx.doi.org/10.1007/s12015-021-10125-xDOI Listing
March 2021

Accelerating innovation and commercialization through standardization of microfluidic-based medical devices.

Lab Chip 2021 01;21(1):9-21

National Institute of Standards and Technology, 100 Bureau Drive, MS8120, Gaithersburg, MD, 20899 USA.

Worldwide, the microfluidics industry has grown steadily over the last 5 years, with the market for microfluidic medical devices experiencing a compound growth rate of 22%. The number of submissions of microfluidic-based devices to regulatory agencies such as the U.S. Food & Drug Administration (FDA) has also steadily increased, creating a strong demand for the development of consistent and accessible tools for evaluating microfluidics-based devices. The microfluidics community has been slow, or even reluctant, to adopt standards and guidelines, which are needed for harmonization and for assisting academia, researchers, designers, and industry across all stages of product development. Appropriate assessments of device performance also remain a bottleneck for microfluidic devices. Standards reside at the core of mature supply chains generating economies of scale and forging a consistent pathway to match stakeholder expectations, thus creating a foundation for successful commercialization. This article provides a unique perspective on the need for the development of standards specific to the emerging biomedical field of microfluidics. Our aim is to facilitate innovation by encouraging the microfluidics community to work together to help bridge knowledge gaps and improve efficiency in getting high-quality microfluidic medical devices to market faster. We start by acknowledging the progress that has been made in various areas over the past decade. We then describe the existing gaps in the standardization of flow control, interconnections, component integration, manufacturing, assembly, packaging, reliability, performance of microfluidic elements and safety testing of microfluidic devices throughout the entire product life cycle.
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http://dx.doi.org/10.1039/d0lc00963fDOI Listing
January 2021

Efficient Development of Integrated Lab-On-A-Chip Systems Featuring Operational Robustness and Manufacturability.

Authors:
Jens Ducrée

Micromachines (Basel) 2019 Dec 17;10(12). Epub 2019 Dec 17.

FPC@DCU-Fraunhofer Project Centre for Embedded Bioanalytical Systems at Dublin City University, School of Physical Sciences, Glasnevin, Dublin 9, Ireland.

The majority of commercially oriented microfluidic technologies provide novel point-of-use solutions for laboratory automation with important areas in the context of the life sciences such as health care, biopharma, veterinary medicine and agrifood as well as for monitoring of the environment, infrastructures and industrial processes. Such systems are often composed of a modular setup exhibiting an instrument accommodating rather conventional actuation, detection and control units which interfaces with a fluidically integrated "Lab-on-a-Chip" device handling (bio-)sample(s) and reagents. As the complex network of tiny channels, chambers and surface-functionalised zones can typically not be properly cleaned and regenerated, these microfluidic chips are mostly devised as single-use disposables. The availability of cost-efficient materials and associated structuring, functionalisation and assembly schemes thus represents a key ingredient along the commercialisation pipeline and will be a first focus of this work. Furthermore, and owing to their innate variability, investigations on biosamples mostly require the acquisition of statistically relevant datasets. Consequently, intermediate numbers of consistently performing chips are already needed during application development; to mitigate the potential pitfalls of technology migration and to facilitate regulatory compliance of the end products, manufacture of such pilot series should widely follow larger-scale production schemes. To expedite and de-risk the development of commercially relevant microfluidic systems towards high Technology Readiness Levels (TRLs), we illustrate a streamlined, manufacturing-centric platform approach employing the paradigms of tolerance-forgiving Design-for-Manufacture (DfM) and Readiness for Scale-up (RfS) from prototyping to intermediate pilot series and eventual mass fabrication. Learning from mature industries, we further propose pursuing a platform approach incorporating aspects of standardisation in terms of specification, design rules and testing methods for materials, components, interfaces, and operational procedures; this coherent strategy will foster the emergence of dedicated commercial supply chains and also improve the economic viability of Lab-on-a-Chip systems often targeting smaller niche markets by synergistically bundling technology development.
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http://dx.doi.org/10.3390/mi10120886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953106PMC
December 2019

Label-Free Multi Parameter Optical Interrogation of Endothelial Activation in Single Cells using a Lab on a Disc Platform.

Sci Rep 2019 03 11;9(1):4157. Epub 2019 Mar 11.

Dublin City University, School of Physical Sciences, National Centre for Sensor Research, Dublin, Ireland.

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.
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http://dx.doi.org/10.1038/s41598-019-40612-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411894PMC
March 2019

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

Biosens Bioelectron 2018 Nov 29;119:86-93. Epub 2018 Jul 29.

FPC@DCU - Fraunhofer Project Centre for Embedded Bioanalytical Systems at Dublin City University, School of Physical Sciences, Dublin City University, Ireland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bios.2018.07.056DOI Listing
November 2018

Novel Microfluidic Analytical Sensing Platform for the Simultaneous Detection of Three Algal Toxins in Water.

ACS Omega 2018 Jun 20;3(6):6624-6634. Epub 2018 Jun 20.

School of Chemical Sciences, DCU Water Institute, School of Biotechnology, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.

Globally, the need for "on-site" algal-toxin monitoring has become increasingly urgent due to the amplified demand for fresh-water and for safe, "toxin-free" shellfish and fish stocks. Herein, we describe the first reported, Lab-On-A-Disc (LOAD) based-platform developed to detect microcystin levels in situ, with initial detectability of saxitoxin and domoic acid also reported. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device capable of multianalyte sampling. A low-complexity "LED-photodiode" based optical sensing system was tailor-made for the platform, which allows the fluorescence signal of the toxin-specific reaction to be quantified. This system can rapidly and accurately detect the presence of microcystin-LR, domoic acid, and saxitoxin in 30 min, with a minimum of less than 5 min end-user interaction for maximum reproducibility. This method provides a robust "point of need" diagnostic alternative to the current laborious and costly methods used for qualitative toxin monitoring.
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http://dx.doi.org/10.1021/acsomega.8b00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045346PMC
June 2018

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance.

Biotechnol J 2019 Mar 18;14(3):e1800219. Epub 2018 Jul 18.

National Institute for Cellular Biotechnology, Dublin City University, D09 NR58, Dublin, Ireland.

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.
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http://dx.doi.org/10.1002/biot.201800219DOI Listing
March 2019

Wirelessly powered and remotely controlled valve-array for highly multiplexed analytical assay automation on a centrifugal microfluidic platform.

Biosens Bioelectron 2018 Jun 10;109:214-223. Epub 2018 Mar 10.

Institute of Microstructure Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany.

In this paper we present a wirelessly powered array of 128 centrifugo-pneumatic valves that can be thermally actuated on demand during spinning. The valves can either be triggered by a predefined protocol, wireless signal transmission via Bluetooth, or in response to a sensor monitoring a parameter like the temperature, or homogeneity of the dispersion. Upon activation of a resistive heater, a low-melting membrane (Parafilm™) is removed to vent an entrapped gas pocket, thus letting the incoming liquid wet an intermediate dissolvable film and thereby open the valve. The proposed system allows up to 12 heaters to be activated in parallel, with a response time below 3 s, potentially resulting in 128 actuated valves in under 30 s. We demonstrate, with three examples of common and standard procedures, how the proposed technology could become a powerful tool for implementing diagnostic assays on Lab-on-a-Disc. First, we implement wireless actuation of 64 valves during rotation in a freely programmable sequence, or upon user input in real time. Then, we show a closed-loop centrifugal flow control sequence for which the state of mixing of reagents, evaluated from stroboscopically recorded images, triggers the opening of the valves. In our last experiment, valving and closed-loop control are used to facilitate centrifugal processing of whole blood.
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http://dx.doi.org/10.1016/j.bios.2018.03.012DOI Listing
June 2018

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

PLoS One 2017 21;12(12):e0189923. Epub 2017 Dec 21.

FPC@DCU, Fraunhofer Project Centre at Dublin City University, Dublin City University, Glasnevin, Dublin 9, Ireland.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189923PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739450PMC
January 2018

Polyethylene imine/graphene oxide layer-by-layer surface functionalization for significantly improved limit of detection and binding kinetics of immunoassays on acrylate surfaces.

Colloids Surf B Biointerfaces 2017 Oct 27;158:167-174. Epub 2017 Jun 27.

National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address:

Antibody immobilization on polymeric substrates is a key manufacturing step for microfluidic devices that implement sample-to-answer automation of immunoassays. In this work, a simple and versatile method to bio-functionalize poly(methylmethacrylate) (PMMA), a common material of such "Lab-on-a-Chip" systems, is proposed; using the Layer-by-Layer (LbL) technique, we assemble nanostructured thin films of poly(ethylene imine) (PEI) and graphene oxide (GO). The wettability of PMMA surfaces was significantly augmented by the surface treatment with (PEI/GO) film, with an 81% reduction of the contact angle, while the surface roughness increased by 600%, thus clearly enhancing wettability and antibody binding capacity. When applied to enzyme-linked immunosorbent assays (ELISAs), the limit of detection of PMMA surface was notably improved from 340pgmL on commercial grade polystyrene (PS) and 230pgmL on plain PMMA surfaces to 130pgmL on (PEI/GO) treated PMMA. Furthermore, the accelerated antibody adsorption kinetics on the LbL films of GO allowed to substantially shorten incubation times, e.g. for anti-rat IgG adsorption from 2h down to 15min on conventional and treated surfaces, respectively.
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http://dx.doi.org/10.1016/j.colsurfb.2017.06.042DOI Listing
October 2017

Fully automated chemiluminescence detection using an electrified-Lab-on-a-Disc (eLoaD) platform.

Lab Chip 2016 10;16(20):4002-4011

Institute of Microstructure Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany.

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.
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http://dx.doi.org/10.1039/c6lc00973eDOI Listing
October 2016

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

Micromachines (Basel) 2016 Oct 1;7(10). Epub 2016 Oct 1.

School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.

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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http://dx.doi.org/10.3390/mi7100175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189914PMC
October 2016

Xurography actuated valving for centrifugal flow control.

Lab Chip 2016 09 15;16(18):3454-9. Epub 2016 Aug 15.

Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland.

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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http://dx.doi.org/10.1039/c6lc00568cDOI Listing
September 2016

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

PLoS One 2016 11;11(5):e0155545. Epub 2016 May 11.

School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155545PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864222PMC
July 2017

Phase-selective graphene oxide membranes for advanced microfluidic flow control.

Microsyst Nanoeng 2016 11;2:16008. Epub 2016 Apr 11.

School of Physical Sciences, Dublin City University, Dublin 9, Ireland.

For the first time, we harness the unique phase-selectivity of chip-integrated graphene oxide (GO) membranes to significantly enhance flow control on centrifugal microfluidic platforms. In this paper, we present novel processes for the assembly of these GO membranes into polymeric microfluidic systems and demonstrate that multilayer GO membranes allow the passage of water while blocking pressurized air and organic solutions.
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http://dx.doi.org/10.1038/micronano.2016.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444712PMC
April 2016

CD-Based Microfluidics for Primary Care in Extreme Point-of-Care Settings.

Micromachines (Basel) 2016 Jan 29;7(2). Epub 2016 Jan 29.

Institute of Microstructure Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany.

We review the utility of centrifugal microfluidic technologies applied to point-of-care diagnosis in extremely under-resourced environments. The various challenges faced in these settings are showcased, using areas in India and Africa as examples. Measures for the ability of integrated devices to effectively address point-of-care challenges are highlighted, and centrifugal, often termed CD-based microfluidic technologies, technologies are presented as a promising platform to address these challenges. We describe the advantages of centrifugal liquid handling, as well as the ability of a standard CD player to perform a number of common laboratory tests, fulfilling the role of an integrated lab-on-a-CD. Innovative centrifugal approaches for point-of-care in extremely resource-poor settings are highlighted, including sensing and detection strategies, smart power sources and biomimetic inspiration for environmental control. The evolution of centrifugal microfluidics, along with examples of commercial and advanced prototype centrifugal microfluidic systems, is presented, illustrating the success of deployment at the point-of-care. A close fit of emerging centrifugal systems to address a critical panel of tests for under-resourced clinic settings, formulated by medical experts, is demonstrated. This emphasizes the potential of centrifugal microfluidic technologies to be applied effectively to extremely challenging point-of-care scenarios and in playing a role in improving primary care in resource-limited settings across the developing world.
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http://dx.doi.org/10.3390/mi7020022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190444PMC
January 2016

Rapid, culture-independent, optical diagnostics of centrifugally captured bacteria from urine samples.

Biomicrofluidics 2015 Jul 11;9(4):044118. Epub 2015 Aug 11.

Biomedical Diagnostics Institute, National Centre of Sensor Research, School of Physical Sciences, Dublin City University , Glasnevin, Dublin 9, Ireland.

This work presents a polymeric centrifugal microfluidic platform for the rapid and sensitive identification of bacteria directly from urine, thus eliminating time-consuming cultivation steps. This "Lab-on-a-Disc" platform utilizes the rotationally induced centrifugal field to efficiently capture bacteria directly from suspension within a glass-polymer hybrid chip. Once trapped in an array of small V-shaped structures, the bacteria are readily available for spectroscopic characterization, such as Raman spectroscopic fingerprinting, providing valuable information on the characteristics of the captured bacteria. Utilising fluorescence microscopy, quantification of the bacterial load has been achieved for concentrations above 2 × 10(-7) cells ml(-1) within a 4 μl sample. As a pilot application, we characterize urine samples from patients with urinary tract infections. Following minimal sample preparation, Raman spectra of the bacteria are recorded following centrifugal capture in stopped-flow sedimentation mode. Utilizing advanced analysis algorithms, including extended multiplicative scattering correction, high-quality Raman spectra of different pathogens, such as Escherichia coli or Enterococcus faecalis, are obtained from the analyzed patient samples. The whole procedure, including sample preparation, requires about 1 h to obtain a valuable result, marking a significant reduction in diagnosis time when compared to the 24 h and more typically required for standard microbiological methods. As this cost-efficient centrifugal cartridge can be operated using low-complexity, widely automated instrumentation, while providing valuable bacterial identification in urine samples in a greatly reduced time-period, our opto-microfluidic Lab-on-a-Disc device demonstrates great potential for next-generation patient diagnostics at the of point-of-care.
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http://dx.doi.org/10.1063/1.4928070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537478PMC
July 2015

Real-time monitoring of cell migration, phagocytosis and cell surface receptor dynamics using a novel, live-cell opto-microfluidic technique.

Anal Chim Acta 2015 May 26;872:95-9. Epub 2014 Dec 26.

Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland; School of Physical Sciences, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address:

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell-cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective-diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.
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http://dx.doi.org/10.1016/j.aca.2014.12.035DOI Listing
May 2015

Label-free impedance detection of cancer cells from whole blood on an integrated centrifugal microfluidic platform.

Biosens Bioelectron 2015 Jun 30;68:382-389. Epub 2014 Dec 30.

Biomedical Diagnostics Institute, National Center for Sensor Research, Dublin City University, Ireland; School of Physical Sciences, Dublin City University, Ireland. Electronic address:

An electrochemical Lab-on-a-Disc (eLoaD) platform for the automated quantification of ovarian cancer cells (SKOV3) from whole blood is reported. This centrifugal microfluidic system combines complex sample handling, i.e., blood separation and cancer cell extraction from plasma, with specific capture and sensitive detection using label-free electrochemical impedance. Flow control is facilitated using rotationally actuated valving strategies including siphoning, capillary and centrifugo-pneumatic dissolvable-film (DF) valves. For the detection systems, the thiol-containing amino acid, L-Cysteine, was self-assembled onto smooth gold electrodes and functionalized with anti-EpCAM. By adjusting the concentration of buffer electrolyte, the thickness of the electrical double layer was extended so the interfacial electric field interacts with the bound cells. Significant impedance changes were recorded at 117.2 Hz and 46.5 Hz upon cell capture. Applying AC amplitude of 50 mV at 117.2 Hz and open circuit potential, a minimum of 214 captured cells/mm(2) and 87% capture efficiency could be recorded. The eLoaD platform can perform five different assays in parallel with linear dynamic range between 16,400 and (2.6±0.0003)×10(6) cancer cells/mL of blood, i.e. covering nearly three orders of magnitude. Using the electrode area of 15.3 mm(2) and an SKOV3 cell radius of 5 µm, the lower detection limit is equivalent to a fractional surface coverage of approximately 2%, thus making eLoaD a highly sensitive and efficient prognostic tool that can be developed for clinical settings where ease of handling and minimal sample preparation are paramount.
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http://dx.doi.org/10.1016/j.bios.2014.12.049DOI Listing
June 2015

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

Cytometry A 2015 Jan 12;87(1):74-80. Epub 2014 Nov 12.

Biomedical Diagnostics Institute, National Centre of Sensor Research, School of Physical Sciences, Dublin City University, Dublin, Ireland.

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.
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http://dx.doi.org/10.1002/cyto.a.22588DOI Listing
January 2015

Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

Lab Chip 2014 Sep 31;14(18):3629-39. Epub 2014 Jul 31.

School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.
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http://dx.doi.org/10.1039/c4lc00618fDOI Listing
September 2014

Rapid, low-cost and instrument-free CD4+ cell counting for HIV diagnostics in resource-poor settings.

Lab Chip 2014 Aug 9;14(15):2844-51. Epub 2014 Jun 9.

Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Ireland.

We present a novel, user-friendly and widely autonomous point-of-care diagnostic to enable HIV monitoring in resource-poor regions where the current pandemic is most prevalent. To specifically isolate magnetically tagged CD4+ cells directly from patient blood, the low-cost and disposable microfluidic chip operates by dual-force CD4+ cell magnetophoresis; whereby the interplay of flow and magnetic fields governs the trajectory of target cells depending on whether the cell binds to a magnetic microbead. Instrument-free pumping is implemented by a finger-actuated elastic membrane; tagged beads are laterally deflected by a small and re-useable permanent magnet. The single-depth and monolithic microfluidic structure can easily be fabricated in a single casting step. After their magnetophoretic isolation from whole blood, estimation of CD4+ cell concentrations is then measured by bright-field inspection of the capture chamber. In addition, an optional fluorescence measurement can be used for confirmation of the bright-field result if required. On-chip CD4+ estimation produces a linear response over the full range of medically relevant CD4+ cell concentrations. Our technology combines high-efficiency capture (93.0 ± 3.3%) and cell enumeration.
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http://dx.doi.org/10.1039/c4lc00264dDOI Listing
August 2014

Event-triggered logical flow control for comprehensive process integration of multi-step assays on centrifugal microfluidic platforms.

Lab Chip 2014 Jul 9;14(13):2249-58. Epub 2014 May 9.

Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland.

The centrifugal "lab-on-a-disc" concept has proven to have great potential for process integration of bioanalytical assays, in particular where ease-of-use, ruggedness, portability, fast turn-around time and cost efficiency are of paramount importance. Yet, as all liquids residing on the disc are exposed to the same centrifugal field, an inherent challenge of these systems remains the automation of multi-step, multi-liquid sample processing and subsequent detection. In order to orchestrate the underlying bioanalytical protocols, an ample palette of rotationally and externally actuated valving schemes has been developed. While excelling with the level of flow control, externally actuated valves require interaction with peripheral instrumentation, thus compromising the conceptual simplicity of the centrifugal platform. In turn, for rotationally controlled schemes, such as common capillary burst valves, typical manufacturing tolerances tend to limit the number of consecutive laboratory unit operations (LUOs) that can be automated on a single disc. In this paper, a major advancement on recently established dissolvable film (DF) valving is presented; for the very first time, a liquid handling sequence can be controlled in response to completion of preceding liquid transfer event, i.e. completely independent of external stimulus or changes in speed of disc rotation. The basic, event-triggered valve configuration is further adapted to leverage conditional, large-scale process integration. First, we demonstrate a fluidic network on a disc encompassing 10 discrete valving steps including logical relationships such as an AND-conditional as well as serial and parallel flow control. Then we present a disc which is capable of implementing common laboratory unit operations such as metering and selective routing of flows. Finally, as a pilot study, these functions are integrated on a single disc to automate a common, multi-step lab protocol for the extraction of total RNA from mammalian cell homogenate.
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http://dx.doi.org/10.1039/c4lc00380bDOI Listing
July 2014

A portable centrifugal analyser for liver function screening.

Biosens Bioelectron 2014 Jun 27;56:352-8. Epub 2014 Jan 27.

Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Ireland; School of Physical Sciences, Dublin City University, Ireland.

Mortality rates of up to 50% have been reported after liver failure due to drug-induced hepatotoxicity and certain viral infections (Gao et al., 2008). These adverse conditions frequently affect HIV and tuberculosis patients on regular medication in resource-poor settings. Here, we report full integration of sample preparation with the read-out of a 5-parameter liver assay panel (LAP) on a portable, easy-to-use, fast and cost-efficient centrifugal microfluidic analysis system (CMAS). Our unique, dissolvable-film based centrifugo-pneumatic valving was employed to provide sample-to-answer fashion automation for plasma extraction (from finger-prick of blood), metering and aliquoting into separate reaction chambers for parallelized colorimetric quantification during rotation. The entire LAP completes in less than 20 min while using only a tenth the reagent volumes when compared with standard hospital laboratory tests. Accuracy of in-situ liver function screening was validated by 96 separate tests with an average coefficient of variance (CV) of 7.9% compared to benchtop and hospital lab tests. Unpaired two sample statistical t-tests were used to compare the means of CMAS and benchtop reader, on one hand; and CMAS and hospital tests on the other. The results demonstrate no statistical difference between the respective means with 94% and 92% certainty of equivalence, respectively. The portable platform thus saves significant time, labour and costs compared to established technologies, and therefore complies with typical restrictions on lab infrastructure, maintenance, operator skill and costs prevalent in many field clinics of the developing world. It has been successfully deployed to a centralised lab in Nigeria.
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http://dx.doi.org/10.1016/j.bios.2014.01.031DOI Listing
June 2014

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

Biomicrofluidics 2013 8;7(3):34101. Epub 2013 May 8.

School of Physical Sciences, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland ; Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.

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.
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http://dx.doi.org/10.1063/1.4804250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663865PMC
January 2014

Fluidic automation of nitrate and nitrite bioassays in whole blood by dissolvable-film based centrifugo-pneumatic actuation.

Sensors (Basel) 2013 Aug 26;13(9):11336-49. Epub 2013 Aug 26.

Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.

This paper demonstrates the full centrifugal microfluidic integration and automation of all liquid handling steps of a 7-step fluorescence-linked immunosorbent assay (FLISA) for quantifying nitrate and nitrite levels in whole blood within about 15 min. The assay protocol encompasses the extraction of metered plasma, the controlled release of sample and reagents (enzymes, co-factors and fluorescent labels), and incubation and detection steps. Flow control is implemented by a rotationally actuated dissolvable film (DF) valving scheme. In the valves, the burst pressure is primarily determined by the radial position, geometry and volume of the valve chamber and its inlet channel and can thus be individually tuned over an extraordinarily wide range of equivalent spin rates between 1,000 RPM and 5,500 RPM. Furthermore, the vapour barrier properties of the DF valves are investigated in this paper in order to further show the potential for commercially relevant on-board storage of liquid reagents during shelf-life of bioanalytical, ready-to-use discs.
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http://dx.doi.org/10.3390/s130911336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821360PMC
August 2013

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

J Lab Autom 2014 Jun 20;19(3):285-96. Epub 2013 Sep 20.

Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Ireland

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.
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http://dx.doi.org/10.1177/2211068213504759DOI Listing
June 2014

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics.

Anal Chim Acta 2013 Jun 18;781:54-62. Epub 2013 Apr 18.

Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland.

In this paper we report a centrifugal microfluidic "lab-on-a-disc" system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL(-1) of hIgG.
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http://dx.doi.org/10.1016/j.aca.2013.04.016DOI Listing
June 2013

CD4 counting technologies for HIV therapy monitoring in resource-poor settings--state-of-the-art and emerging microtechnologies.

Lab Chip 2013 Jul;13(14):2731-48

Biomedical Diagnostic Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Dublin, Ireland.

Modern advancements in pharmaceuticals have provided individuals who have been infected with the human immunodeficiency virus (HIV) with the possibility of significantly extending their survival rates. When administered sufficiently soon after infection, antiretroviral therapy (ART) allows medical practitioners to control onset of the symptoms of the associated acquired immune deficiency syndrome (AIDS). Active monitoring of the immune system in both HIV patients and individuals who are regarded as "at-risk" is critical in the decision making process for when to start a patient on ART. A reliable and common method for such monitoring is to observe any decline in the number of CD4 expressing T-helper cells in the blood of a patient. However, the technology, expertise, infrastructure and costs to carry out such a diagnostic cannot be handled by medical services in resource-poor regions where HIV is endemic. Addressing this shortfall, commercialized point-of-care (POC) CD4 cell count systems are now available in such regions. A number of newer devices will also soon be on the market, some the result of recent maturing of charity-funded initiatives. Many of the current and imminent devices are enabled by microfluidic solutions, and this review will critically survey and analyze these POC technologies for CD4 counting, both on-market and near-to-market deployment. Additionally, promising technologies under development that may usher in a new generation of devices will be presented.
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http://dx.doi.org/10.1039/c3lc50213aDOI Listing
July 2013