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

Content in the UH Research Archive is made available for personal research, educational, and non-commercial purposes only. Unless otherwise stated, all content is protected by copyright, and in the absence of an open license, permissions for further re-use should be sought from the publisher, the author, or other copyright holder.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.Peer reviewe

Solvent-selective routing for centrifugally automated solid-phase purification of RNA

The final publication is available at Springer via https://doi.org/10.1007/s10404-014-1477-9.We present a disc-based module for rotationally controlled solid-phase purification of RNA from cell lysate. To this end, multi-stage routing of a sequence of aqueous and organic liquids into designated waste and elution reservoirs is implemented by a network of strategically placed, solvent-selective composite valves. Using a bead-based stationary phase at the entrance of the router, we show that total RNA is purified with high integrity from cultured MCF7 and T47D cell lines, human leucocytes and Haemophilus influenzae cell lysates. Furthermore, we demonstrate the broad applicability of the device through the in vitro amplification of RNA purified on-disc using RT-PCR and NASBA. Our novel router will be at the pivot of a forthcoming, fully integrated and automated sample preparation system for RNA-based analysis.Peer reviewe

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

Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs - Fig 7

<p><b>Phase Switching Data and Quantitative Results</b> (a-b) highlight ‘phase-switching’. This trait occurs where the system switches from drawing one phase, the DGM, to the other phase, plasma, while leaving a significant number of PBMCs within the main sedimentation chamber (c) and image from the haemocytometer showing mononuclear leukocytes enumeration (d) comparison of mononuclear leukocytes extracted from the single pneumatic chamber (Disc A) to a whole blood count (hospital laboratory) and using a HemoCue™.</p

Projector mode operation.

<p>(i) Schematic of the reader in projector mode. A shadow of the read chamber can be projected onto a wall from a distance of ~1 m and can easily be discerned in a dim or dark room (ii) an image (acquired using a smartphone) of the read chamber shadow projected onto a wall. This is read as ‘5’ relative to the graduated markings. The projected image is approximately 50 times larger than the read chamber.</p

Comparison of Hydrostatic, Dynamic and Hybrid centrifugo-pneumatic siphon valves (CPSVs).

<p>Gas pressure is indicated in subfigures through the intensity of colour. (a) Liquid is loaded to the disc. (b) Upon spinning, the liquid advances into the central chamber while seeking hydrostatic equilibrium. However, the centrifugal compression of the gas volumes in the compartments enclosed by the liquid creates a counter pressure. (c, d) In the hydrostatic mechanism, the air in the closed side chamber expands upon reduction of the spin rate, so the liquid level in the open central chamber rises above the crest point of the siphon to forward the liquid into the open receiving chamber. In the hybrid CPSV, air is compressed in the closed central chamber during fast spinning. After lowering the angular frequency, the resulting decompression of air and the reduction of the centrifugal field jointly lift the liquid levels in the side arms above the crest point to empty the liquid into the open outer chamber. The operation of the dynamic CPSV follows a similar mechanism. However, the crest point of the siphon is now located above the level of the hydrostatic equilibrium; the siphon valve thus only opens upon rapid change of the spin rate so inertia propels the flow until the meniscus in the outlet channel has protruded past the liquid level in the central chamber.</p

Schematic of Multi-layer discs Multi-layer discs are assembled from of 1.5-mm PMMA layers interspersed by 86-μm thin PSA films.

<p>Schematic of Multi-layer discs Multi-layer discs are assembled from of 1.5-mm PMMA layers interspersed by 86-μm thin PSA films.</p

WBC isolation using a dynamically CPSV (Disc D).

<p>(a) The disc loaded with DGM while the whole blood is introduced during disc acceleration. (b) RBCs sediment to the bottom. Note that the pneumatic chamber is extended by channel (lower level) indicated in a blue dash. This large pneumatic chamber is required to ensure that the valves function at the volumes processed. (c) Stratified blood in the chamber. (d) The spin rate is decreased and both siphons are simultaneously primed. Note that the siphon crests are located radially inwards of the bulk liquid and the liquid displaced radially inwards along the loading channel. (e) The spin rate is increased and both siphons empty. See ESI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155545#pone.0155545.s001" target="_blank">S1 Movie</a> showing blood processing in Disc D.</p

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

<div><p>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.</p></div

Comparison of blood centrifugation in configurations with continuous (Disc A) and split pneumatic chambers (Disc B).

<p>Note that only in the single chamber design (top) RBCs enter the pneumatic chamber due to the increased liquid displacement (c). (d) Note the different PBMC locations in the two designs above and below the siphon outlet. See ESI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155545#pone.0155545.s001" target="_blank">S1 Movie</a> showing blood processing in Disc B.</p