Automated DNA purification and Multiplexed lamp assay preparation On a centrifugal microfluidic “lab-on-a-disc” platform

This work presents a rotational-pulse actuated microfluidic cartridge enabling automated detection of plant pathogens on a compact device towards point-of-use monitoring of food safety. This highly integrated “Lab-on-a-Disc” (LoaD) system first runs the sample over a stationary phase of silica beads, followed by ethanol (EtOH) wash and final elution of DNA. The eluate is then homogenized using ‘shake mode’ agitation, accurately metered and then mixed with reagents for loop-mediated isothermal amplification (LAMP). We successfully purify plant DNA and demonstrate on-disc quantitative LAMP amplification

Nucleic acid purification on a lab-on a-disc with time-controlled incubation

Here we present integrated Lab-on-a-Disc (LoaD) cartridges which are applied to the purification of nucleic acid using the silica bead based method. We utilize a novel combination of ‘event-triggered’ dissolvable film (DF) valving [1] and a centrifugo-pneumatic siphon valve (CPSV) [2] to permit timing of sample incubations, washes and DNA elution. We present two systems; one in with a conventional ‘wash through’ elution which achieves a purification yield of 32.3% ± 5.4% (n = 4). The second system, which uses a CPSV to enable extended wash and elution steps, provides an increased efficiency of 58.4 ± 7.5% (n = 3)

Comparison of early life history of the bay scallop, Argopecten irradians : effects of microalgal diets on growth and biochemical composition

The culture of bay scallops, Argopecten irradians, is limited by a reliable and affordable supply of spat and the ability to ensure that animals attain market size within a single growing season. The main goals of our study were thus: (1) to develop growth-optimizing algal diets for implementation in hatcheries, and (2) to identify and compare bay scallop postlarval and juvenile dietary requirements, especially of lipids and fatty acids, which if met may enhance production. Nutritional needs of postlarval bay scallops (present study) are compared with those of sea scallops, Placopecten magellanicus, offered the same diets in a previous companion study. To this end, postlarval (initial shell height, SH = 240 ?m) and juvenile (initial SH = 10 mm) bay scallops were offered 6\u20137 microalgal diet combinations at 20 \uc2\ub0C, for 3 weeks. A similar growth ranking among diets was observed between the two developmental stages. A combination diet of Pavlova sp. (CCMP 459) and Chaetoceros muelleri was far superior to any other diet tested, yielding growth rates of 58 and 357 ?m day? 1 which were 65% and 25% higher than the next highest performing diet of Tetraselmis striata/C. muelleri in postlarvae and juveniles, respectively. The T. striata/C. muelleri diet, which is limited in the n-3 fatty acid docosahexaenoic acid (DHA), yielded very poor growth of sea scallop postlarvae in a prior study, indicating that bay scallops may have less stringent requirements for DHA than sea scallops. The Pav 459/C. muelleri diet, which also supported the highest growth of sea scallop postlarvae, is characterized by elevated levels of the n-6 fatty acids, arachidonic (AA) in C. muelleri and 4,7,10,13,16-docosapentaenoic (DPA) in Pav 459. The two diets deficient in AA and n-6 DPA, Pavlova lutheri/Thalassiosira weissflogii and P. lutheri/Fragilaria familica, yielded the lowest growth rates in both bay scallop postlarvae and juveniles. Tissue enrichment of these two fatty acids relative to the diet, as well as overall enrichment in ?n-6 fatty acids was observed across developmental stages and dietary treatments. A similar pattern has previously been observed in sea scallop postlarvae, suggesting a dietary requirement for n-6 fatty acids in pectinids that has often been overlooked in the past.Peer reviewed: YesNRC publication: Ye

Programmable fluidic networks on centrifugal microfluidic discs

Background: Biomedical diagnostic and lab automation solutions built on the Lab-on-a-Disc (LoaD) platform has great potential due to their independence from specialized micro-pumps and their ease of integration, through direct pipetting, with manual or automated workflows. However, a challenge for all microfluidic chips is their cost of manufacture when each microfluidic disc must be customized for a specific application. In this paper, we present centrifugal discs with programmable fluidic networks. Results: Based on dissolvable film valves, we present two technologies. The first, based on recently introduced pulse-actuated dissolvable film valves, is a centrifugal disc which, depending on how it is loaded, is configured to perform either six sequential reagent releases through one reaction chamber or three sequential reagent releases through two reaction chambers. In the second approach, we use the previously introduced electronic Lab-on-a-Disc (eLoaD) wireless valve array, which can actuate up to 128 centrifugo-pneumatic dissolvable film valves in a pre-defined sequence. In this approach we present a disc which can deliver any one of 8 reagent washes to any one of four reaction chambers. We use identical discs to demonstrate the first four sequential washes through two reaction chambers and then two sequential washes through four reaction chambers. Significance: These programmable fluidic networks have the potential to allow a single disc architecture to be applied to multiple different assay types and so can offer a lower-cost and more integrated alternative to the standard combination of micro-titre plate and liquid handling robot. Indeed, it may even be possible to conduct multiple different assays concurrently. This can have the effect of reducing manufacturing costs and streamlining supply-chains and so results in a more accessible diagnostic platform

Wireless closed-loop control of centrifugo-pneumatic valving towards large-scale microfluidic process integration

We present for the first time an integrated wirelessly powered Arduino processor and Bluetooth interface that are co-rotated with the cartridge to allow large-scale process integration on a centrifugal microfluidic platform. This highly modular, electronically controlled “Lab-on-aDisc” system (eLoaD) can independently actuate up to 128 normally-closed valves by an array of microheaters during rotation for comprehensive, highly parallelized sample-toanswer automation. Additionally, we implement real-time optical (colour intensity) measurement for closed-loop control of liquid handling, sample preparation and detection

Plant pathogen detection on a lab-on-a-disc using solid-phase extraction and isothermal nucleic acid amplification enabled by digital pulse-actuated dissolvable film valves

By virtue of its ruggedness, portability, rapid processing times, and ease-of-use, academic and commercial interest in centrifugal microfluidic systems has soared over the last decade. A key advantage of the LoaD platform is the ability to automate laboratory unit operations (LUOs) (mixing, metering, washing etc.) to support direct translation of ‘on-bench’ assays to ‘on-chip’. Additionally, the LoaD requires just a low-cost spindle motor rather than specialized and expensive microfluidic pumps. Furthermore, when flow control (valves) is implemented through purely rotational changes in this same spindle motor (rather than using additional support instrumentation), the LoaD offers the potential to be a truly portable, low-cost and accessible platform. Current rotationally controlled valves are typically opened by sequentially increasing the disc spin-rate to a specific opening frequency. However, due lack of manufacturing fidelity these specific opening frequencies are better described as spin frequency ‘bands’. With low-cost motors typically having a maximum spin-rate of 6000 rpm (100 Hz), using this ‘analogue’ approach places a limitation on the number of valves, which can be serially actuated thus limiting the number of LUOs that can be automated. In this work, a novel flow control scheme is presented where the sequence of valve actuation is determined by architecture of the disc while its timing is governed by freely programmable ‘digital’ pulses in its spin profile. This paradigm shift to ‘digital’ flow control enables automation of multi-step assays with high reliability, with full temporal control, and with the number of LUOs theoretically only limited by available space on the disc. We first describe the operational principle of these valves followed by a demonstration of the capability of these valves to automate complex assays by screening tomato leaf samples against plant pathogens. Reagents and lysed sample are loaded on-disc and then, in a fully autonomous fashion using only spindle-motor control, the complete assay is automated. Amplification and fluorescent acquisition take place on a custom spin-stand enabling the generation of real-time LAMP amplification curves using custom software. To prevent environmental contamination, the entire discs are sealed from atmosphere following loading with internal venting channels permitting easy movement of liquids about the disc. The disc was successfully used to detect the presence of thermally inactivated Clavibacter michiganensis. Michiganensis (CMM) bacterial pathogen on tomato leaf samples