Multiplexed Centrifugal Microfluidic System for Dynamic Solid-Phase Purification of Polynucleic Acids Direct from Buccal Swabs.

This report describes the development of a centrifugally controlled microfluidic dynamic solid-phase extraction (dSPE) platform to reliably obtain amplification-ready nucleic acids (NAs) directly from buccal swab cuttings. To our knowledge, this work represents the first centrifugal microdevice for comprehensive preparation of high-purity NAs from raw buccal swab samples. Direct-from-swab cellular lysis was integrated upstream of NA extraction, and automatable laser-controlled on-board microvalving strategies provided the strict spatiotemporal fluidic control required for practical point-of-need use. Solid-phase manipulation during extraction leveraged the application of a bidirectional rotating magnetic field to promote thorough interaction with the sample (e.g., NA capture). We illustrate the broad utility of this technology by establishing downstream compatibility of extracted nucleic acids with three noteworthy assays, namely, the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). The PCR-readiness of the extracted DNA was confirmed by generating short tandem repeat (STR) profiles following multiplexed amplification. With no changes to assay workflow, viral RNA was successfully extracted from contrived (spiked) SARS-CoV-2 swab samples, confirmed by RT-qPCR. Finally, we demonstrate the compatibility of the extracted DNA with LAMP-a technique well suited for point-of-need genetic analysis due to minimal hardware requirements and compatibility with colorimetric readout. We describe an automatable, portable microfluidic platform for the nucleic acid preparation device that could permit practical, in situ use by nontechnical personnel.

Optically-controlled closable microvalves for polymeric centrifugal microfluidic devices.

Microvalving is a pivotal component in many microfluidic lab-on-a-chip platforms and micro-total analysis systems (µTAS). Effective valving is essential for the integration of multiple unit operations, such as, liquid transport, mixing, aliquoting, metering, washing, and fractionation. The ideal microfluidic system integrates numerous, sequential unit operations, provides precise spaciotemporal reagent release and flow control, and is amenable to rapid, low-cost fabrication and prototyping. Centrifugal microfluidics is an attractive approach that minimizes the need for supporting peripheral hardware. However, many of the microfluidic valving methods described in the literature suffer from operational limitations and fail when high rotational frequencies or pressure heads are required early in the analytical process. Current approaches to valve closure add unnecessary complexity to the microfluidic architecture, require the incorporation of additional materials such as wax, and entail extra fabrication steps or processes. Herein we report the characterization and optimization of a laser-actuated, closable valve method for polymeric microfluidic devices that ameliorates these shortcomings. Under typical operational conditions (rcf ≤605 ×g) a success rate >99% was observed, i.e. successful valve closures remained leak free through 605 ×g. Implementation of the laser-actuated closable valving system is demonstrated on an automated, centrifugally driven dynamic solid phase extraction (dSPE) device. Compatibility of this laser-actuated valve closure approach with commercially available polymerase chain reaction (PCR) assays is established by the generation of full 18-plex STR profiles from DNA purified via on-disc dSPE. This novel approach promises to simplify microscale valving, improve functionality by increasing the number of integrated unit operations, and allow for the automation of progressively complex biochemical assays.