Digital process control of multi-step assays on centrifugal platforms using high-low-high rotational-pulse triggered valving.

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.

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

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 Iκß. 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.

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.

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

Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic "Lab-on-a-Disc" cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are 'low-pass', i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both 'hydrostatically' and 'hydrodynamically' triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, 'dual siphon' configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.

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

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

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.

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

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.

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

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.

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.

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

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.

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.

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.

Centromeres: assembling and propagating epigenetic function.

The faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an epigenetically inherited locus, the centromere, and its role in the processes by which sister chromatids are evenly segregated to daughter cells. During the G2 phase of the cell cycle kinetochores are assembled upon the chromatids. During mitosis, kinetochores attach chromosome(s) to the mitotic spindle. The kinetochore structure serves as the interface between the mitotic spindle and the chromatids and it is at the kinetochore where the forces that drive chromatid separation are generated. Unattached chromosomes fail to satisfy the spindle assembly checkpoint (SAC), resulting in cell cycle arrest. The centromere is the locus upon which the kinetochore assembles, and centromeres themselves are determined by their unique protein composition. Apart from budding yeast, centromeres are not specified simply by DNA sequence, but rather through chromatin composition and architecture and are thus epigenetically determined. Centromeres are built on a specific nucleosome not found elsewhere in the genome, in which histone H3 is replaced with a homologue - CENP-A or CenH3. This domain is flanked by heterochromatin and is folded to provide a 3-dimensional cylinder-like structure at metaphase that establishes the kinetochore on the surface of the mitotic chromosomes. A large family of CENtromere Proteins (CENPs) associates with centromeric chromatin throughout the cell cycle and are required for kinetochore function. Unlike the bulk of histones, CENP-A is not assembled concurrently with DNA synthesis in S-phase but rather assembles into the centromere in the subsequent G1 phase. The assembly of CENP-A chromatin following DNA replication and the re-establishment of this network of constitutive proteins have emerged as critical mechanisms for understanding how the centromere is replicated during the cell cycle.

Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin.

The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin. Poleta-deficient cells are more sensitive to both drugs than are normal cells. In poleta-deficient cells, drug treatment leads to prolonged S-phase arrest, and increased phosphorylation of the phosphatidylinositol-3-kinase-related protein kinase (PIKK) substrates Chk1, p95/Nbs1 and RPA2, the 34kDa subunit of replication protein A. Cisplatin- and oxaliplatin-induced hyperphosphorylation of RPA2, and association of the hyperphosphorylated protein with chromatin, is elevated in poleta-deficient cells. Cisplatin-induced phosphorylation of RPA2 on serine 4/serine 8, but not on serine 33, is inhibited by the DNA-PK inhibitor, NU7441, but not by the ATM inhibitor, KU-55933. Cisplatin-induced DNA-PK-dependent hyperphosphorylation of RPA2 on serine 4/serine 8 occurs after recruitment of RPA to chromatin, as determined by immunofluorescence and by subcellular fractionation. ATR is required both for recruitment of RPA2 to chromatin and its subsequent hyperphosphorylation on serine 4/serine 8 by DNA-PK, since CGK733, an inhibitor of ATM and ATR, blocked both recruitment and hyperphosphorylation. Thus, increased sensitivity to cisplatin and oxaliplatin in DNA poleta-deficient cells is associated with prolonged S-phase arrest, and enhanced PIKK-signalling, in particular activation of DNA-PK-dependent hyperphosphorylation of RPA2 on serines 4 and 8.

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK.

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.