Characterization of a Centrifugal Microfluidic Orthogonal Flow Platform.

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.

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.

Closable Valves and Channels for Polymeric Microfluidic Devices.

This study explores three unique approaches for closing valves and channels within microfluidic systems, specifically multilayer, centrifugally driven polymeric devices. Precise control over the cessation of liquid movement is achieved through either the introduction of expanding polyurethane foam, the application of direct contact heating, or the redeposition of xerographic toner via chloroform solvation and evaporation. Each of these techniques modifies the substrate of the microdevice in a different way. All three are effective at closing a previously open fluidic pathway after a desired unit operation has taken place, i.e., sample metering, chemical reaction, or analytical measurement. Closing previously open valves and channels imparts stringent fluidic control-preventing backflow, maintaining pressurized chambers within the microdevice, and facilitating sample fractionation without cross-contamination. As such, a variety of microfluidic bioanalytical systems would benefit from the integration of these valving approaches.

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.

Color manipulation through microchip tinting for colorimetric detection using hue image analysis.

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-1.