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

Mishra, Rohit; Julius, Lourdes Albina Nirupa; Condon, Jack; Pavelskopfa, Patricija; Early, Philip L.; Dorrian, Matthew; Mrvova, Katarina; Henihan, Grace; Mangwanya, Faith; Dreo, Tanya; Ducrée, Jens; Macdonald, Niall P.; Schoen, C.D.; Kinahan, David J.

doi:10.1016/j.aca.2023.341070

Cited by 7 publications

(22 citation statements)

References 87 publications

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“…78 The benchtop and on-disc protocols have also been described previously. 78 Briefly, reactions of 25 μL volumes were created from primers (4 μL) and LAMP reagent ISO-100 (Optigene, U.K.; 15 μL). The total volume was reached through the addition of "samples" of either 6 μL of buffer containing DNA (for positive controls) or 6 μL of nuclease-free water (for negative controls).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…This permits videos to be created for flow visualization purposes where the disc is rotating but appears stationary. The basic test-stand is further modified with a heating system and a fluorescence detection system as described previously . The instrument is controlled using a custom program (written in LabVIEW, National Instruments, Texas, U.S.A.), which has full control of the instrument, including the heating system (temperature, clamping, unclamping), the disc (speed, acceleration, positioning), and fluorescence detector (laser on/off, acquire measurement).…”

Section: Methodsmentioning

confidence: 99%

“…Before testing, the solid-phase (acid washed glass beads, (Sigma-Aldrich)) is added to the disc using the method described previously. , The end of a standard 1000 μL plastic pipet tip is trimmed to make the opening larger and make it into a funnel. It is aligned with an opening in the disc and filled with beads; the pipet tip is tapped gently, and the beads are gravity fed into the disc.…”

Section: Methodsmentioning

confidence: 99%

“…Plant Sample Preparation. The tomato leaves are prepared using the DNEasy Plant Mini Kit (Qiagen) using a protocol adapted from Mishra et al 78 Briefly, 70 mg of plant material is cooled in liquid nitrogen and ground to powder using a mortar and pestle. At this point, if the sample is spiked with thermally inactivated CMM bacteria (IPO-3208), a 53 μL volume (diluted to the appropriate concentration) is added.…”

Section: Acs Omegamentioning

confidence: 99%

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Low–High–Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc

Julius,

Saeed,

Kuijpers

et al. 2024

ACS Omega

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The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering, mixing, and aliquoting supports on-disc automation of bioassay without the need for extensive biological optimization. Thus, well-established bioassays, normally conducted manually using pipettes or using liquid handling robots, can be relatively easily automated in self-contained microfluidic chips suitable for use in point-of-care or point-of-use settings. The LoaD's ease of automation is largely dependent on valves that can control liquid movement on the rotating disc. The optimum valving strategy for a true low-cost and portable device is rotationally actuated valves, which are actuated by changes in the disc spin-speed. However, due to tolerances in disc manufacturing and variations in reagent properties, most of these valving technologies have inherent variation in their actuation spin-speed. Most valves are actuated through stepped increases in disc spin-speed until the motor reaches its maximum speed (rarely more than 6000 rpm). These manufacturing tolerances combined with this "analogue" mechanism of valve actuation limits the number of LUOs that can be placed on-disc. In this work, we present a novel valving mechanism called low−high−low serial dissolvable film (DF) valves. In these valves, a DF membrane is placed in a dead-end pneumatic chamber. Below an actuation spinspeed, the trapped air prevents liquid wetting and dissolving the membrane. Above this spin-speed, the liquid will enter and wet the DF and open the valve. However, as DFs take ∼40 s to dissolve, the membrane can be wetted, and the disc spin-speed reduced before the film opens. Thus, by placing valves in a series, we can govern on which "digital pulse" in spin-speeding a reagent is released; a reservoir with one serial valve will open on the first pulse, a reservoir with two serial valves on the second, and so on. This "digital" flow control mechanism allows the automation of complex assays with high reliability. In this work, we first describe the operation of the valves, outline the theoretical basis for their operation, and support this analysis with an experiment. Next, we demonstrate how these valves can be used to automate the solid-phase extraction of DNA on on-disc LAMP amplification for applications in plant pathogen detection. The disc was successfully used to extract and detect, from a sample lysed off-disc, DNA indicating the presence of thermally inactivated Clavibacter michiganensis ssp. michiganensis (Cmm), a bacterial pathogen on tomato leaf samples.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Acs Omegamentioning

confidence: 99%

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Low–High–Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc

Julius,

Saeed,

Kuijpers

et al. 2024

ACS Omega

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“…The Centrifugal Disc (CD) or Lab On Disc (LOD) is an integrated platform that utilizes various functional units, including pumps, 1,2 valves, 3 and mixers. 4,5 It enables the execution of rapid, precise, and cost-effective parallel testing, specifically designed for a wide range of biomedical applications, 6 including RBC antigen typing, 7 nucleic acid extraction, 8,9 cell isolation, 10 as well as plasma extraction.…”

Section: Introductionmentioning

confidence: 99%

Real-time monitoring and actuation of a hybrid siphon valve for hematocrit-independent plasma separation from whole blood

Khodadadi,

Pishbin,

Eghbal

et al. 2023

Analyst

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Automated solid phase DNA extraction on a lab-on-a-disc with two-degrees of freedom instrumentation

Carthy,

Hughes,

Higgins

et al. 2023

Analytica Chimica Acta

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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

Cited by 7 publications

References 87 publications

Low–High–Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc

Low–High–Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc

Real-time monitoring and actuation of a hybrid siphon valve for hematocrit-independent plasma separation from whole blood

Automated solid phase DNA extraction on a lab-on-a-disc with two-degrees of freedom instrumentation

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