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

Abstract

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.