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 LAMPa 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.
Colorimetry with microfluidic devices has been proven to be an advantageous method for in situ analyses where limited resources and rapid response for untrained users are desired. Image analysis using a small camera or cell phone can be easily incorporated for an objective readout, eliminating variations from normal differences in color perception and environmental factors during analysis. The image analysis using the parameter hue, for example, has been utilized as a highly effective, objective analysis method that correlates with the psychological way color is perceived. Hue analysis, however, is best used for colorimetric reactions that result in distinct changes from one color to a markedly different color and can be inadequate to distinguish between subtle or monotonal (colorless-to-colored) color changes. We address this with three unique color manipulation (i.e., tinting) techniques that provide greater discrimination with such color changes, thus yielding improved limits of detection for various colorimetric reactions that may have previously been limited. Tinting is invoked through dyeing the reagent substrate, colored printing the device, or colored lighting during image capture, and is shown to effectively shift the background color of the reaction detection area. Hydrogen peroxide, a constituent of peroxide-based explosives, is associated with a monochromatic color change upon reaction, and this is used to demonstrate the effectiveness of the tinting methods in improving the limit of detection from an undetectable color change to 0.1 mg mL.
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