No abstract

Microvalving is a pivotal component in many microfluidic lab-on-a-chip platforms and micro-total analysis systems (μTAS).

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.

To bring to bear the power of centrifugal microfluidics on vertical flow immunoassays, control of flow orthogonally through nanoporous membranes is essential. The on-disc approach described here leverages the rapid print-cut-laminate (PCL) disc fabrication and prototyping method to create a permanent seal between disc materials and embedded nanoporous membranes. Rotational forces drive fluid flow, replacing capillary action, and complex pneumatic pumping systems. Adjacent microfluidic features form a flow path that directs fluid orthogonally (vertically) through these embedded membranes during assay execution. This method for membrane incorporation circumvents the need for solvents (e.g., acetone) to create the membrane-disc bond and sidesteps issues related to undesirable bypass flow. In other recently published work, we described an orthogonal flow (OF) platform that exploited embedded membranes for automation of enzyme-linked immunosorbent assays (ELISAs). Here, we more fully characterize flow patterns and cellulosic membrane behavior within the centrifugal orthogonal flow (cOF) format. Specifically, high-speed videography studies demonstrate that sample volume, membrane pore size, and ionic composition of the sample matrix significantly impact membrane behavior, and consequently fluid drainage profiles, especially when cellulosic membranes are used. Finally, prototype discs are used to demonstrate proof-of-principle for sandwich-type antigen capture and immunodetection within the cOF system.