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

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 spin-speed, 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.

Figure S1: LAMP Amplification Curves from benchtop control experiments.Samples were prepared as per the paper but purified on-bench using Qiagen spin-columns.Then 20 µL LAMP samples were prepared and amplified in a commercial Qiagen Rotor-gene PCR machine with measurements every 30 s. Plant / SOL COX and Plant + 10 7 CFU CMM / SOL COX amplified after ~ 12 minutes while Plant + 10 7 CFU CMM / CMM amplified after ~ 16 minutes.As expected, Plant / CMM did not amplify.Note that earlier on-bench work for this paper was previously presented 1 .
Figure S2: Image of the thermal blocks which clamp the disc top and bottom to heat it to temperature.Note a thermocouple embedded in a liquid filled reservoir can be seen in this image.Figures S3-S5 were measured using this thermocouple.Readers are referred to previous work which more fully describes the disc operation 1,2        Experiments were run using custom control software (LabVIEW) which ran on human editable / readable script files to control the motors, heaters, and fluorescence detectors during amplification.Generally, the user monitored the disc during the assay steps (using a stereoscopically coupled camara) and manually entered the spin-protocol defined in Figure

Figure S3 :
Figure S3: Temperature data indicating the response of temperature in a well to the discs clamping.It takes ~3.5 minutes for the samples to reach temperature.However, a 7-minute delay is used during experiments before the first fluorescence measurement.Note these measurements were made at 70 °C but LAMP amplification took place at 65 °C.Note in this case the thermal blocks were pre-heated prior to clamping.Figure adapted from Kinahan et al 1

Figure S5 :
Figure S5: The time taken for the blocks to reach temperature.This takes ~17 minutes and in view of this delay the on-disc experiments took place with the discs pre-heated to the set-point (ensuring that the reagents in the disc reached temperature in ~3.5 minutes as shown in Figure S3.Figure adapted from Kinahan et al 1 Figure S5: The time taken for the blocks to reach temperature.This takes ~17 minutes and in view of this delay the on-disc experiments took place with the discs pre-heated to the set-point (ensuring that the reagents in the disc reached temperature in ~3.5 minutes as shown in Figure S3.Figure adapted from Kinahan et al 1

Figure S6 :
Figure S6: Image of the fluorescence detection system mounted above the disc.Figure adapted from Kinahan et al 1

Figure S7 :
Figure S7: Schematic of the fluorescence detection configuration.Briefly, a low-cost 460nm is expanded and directed via a dichroic mirror and 10x microscope lens to the sample.It is defocussed when it illuminates the sample resulting in a largely diffuse and uniform excitation light field.Fluorescence emitted by the sample is focussed by the same 10x microscope and directed through the dichroic lens and then focussed onto an amplified detector photodiode.Figure adapted from Kinahan et al 1

Figure S8 :
Figure S8: Calibration data for the fluorescence sensor.First, amplified, and unamplified LAMP reagent, and a serial dilution of fluorescein fluorescence dye were measured in a commercial PCR thermocycler (Qiagen Rotorgene)(left vertical axis).Fluorescent dye was tested in the same concentration using the fluorescence detector and the centrifugal disc (right vertical axis).The axes were scaled to align the fluorescein fluorescence dye measurements to a best fit.Results indicate that the photodiode demonstrates greater linearity and can detect and discriminate fluorescence signals lower than background (unamplified LAMP).Not that actual on-disc samples had signals from 160mV (NTC / unamplified) to 300mV (amplified) which likely indicates slight misalignment of the fluorescence detector compared to the results presented here.Figure adapted from Kinahan et al 1 . Figure adapted from Kinahan et al 1