Digital image analysis for biothreat detection via rapid centrifugal microfluidic orthogonal flow immunocapture.

We report clear proof-of-principle for centrifugally-driven, multiplexed, paper-based orthogonal flow sandwich-style immunocapture (cOFI) and colorimetric detection of Zaire Ebola virus-like particles. Capture antibodies are immobilized onto nanoporous nitrocellulose membranes that are then laminated into polymeric microfluidic discs to yield ready-to-use analytical devices. Fluid flow is controlled solely by rotational speed, obviating the need for complex pneumatic pumping systems, and providing more precise flow control than with the capillary-driven flow used in traditional lateral flow immunoassays (LFIs). Samples containing the antigen of interest and gold nanoparticle-labeled detection antibodies are pumped centrifugally through the embedded, prefunctionalized membrane where they are subsequently captured to generate a positive, colorimetric signal. When compared to the equivalent LFI counterparts, this cOFI approach generated immunochromatographic colorimetric responses that are objectively darker (saturation), more intense (grayscale), and less variable regarding total area of the color response. We also describe an image analysis approach that enables access to rich color data and area statistics without the need for a commercial 'strip reader' or custom-written image analysis algorithms. Instead, our analytical method exploits inexpensive equipment (e.g., smart phone, flatbed scanner, etc.) and freely available software (Fiji distribution of ImageJ) to permit characterization of immunochromatographic responses that includes multiple color metrics, offering insights beyond typical grayscale analysis. The findings reported here stand as clear proof-of-principle for the feasibility of disc-based, centrifugally driven orthogonal flow through a membrane with immunocapture (cOFI) and colorimetric readout of a sandwich-type immunoassay in less than 15 minutes. Once fully developed, this cOFI platform could render a faster, more accurate diagnosis, while processing multiple samples simul-taneously.

A novel method for inward fluid displacement in centrifugal microdevices for highly integrated nucleic acid processing with long-term reagent storage.

Rotationally-driven lab-on-a-disc (LoaD) microfluidic systems are among the most promising methods for realizing complex nucleic acid (NA) testing at the point-of-need (PoN). However, despite significant advancements in NA amplification methods, very few sample-to-answer centrifugal microfluidic platforms have been realized due, in part, to a lack of on-disc sample preparation. In many instances, NA extraction (NAE) and/or lysis must be performed off-disc using conventional laboratory equipment and methods, thus tethering the assay to centralized facilities. Omission of in-line cellular lysis and NAE can be partially attributed to the nature of centrifugally-driven fluidics. Since flow is directed radially outward relative to the center of rotation (CoR), the number of possible sequential unit operations is limited by the disc radius. To address this, we report a simple, practical, automatable, and easy-to-implement method for inward fluid displacement (IFD) compatible with downstream nucleic acid amplification tests (NAATs). This approach leverages carbon dioxide (CO2) gas generated from on-board acid-base neutralization to drive liquid from the disc periphery towards the CoR. Large architectural features or highly corrosive chemicals required in other approaches were replaced with safe-to-handle IFD reagents that maintained their reactivity for at least six months of storage on-disc. Further, spatiotemporal control over neutralization initiation and containment of the resultant pneumatic pressure head was reliably achieved using a single diode for both laser-actuated valve opening and channel sealing, which eliminated the need for manual intervention (e.g., taping over vents) required in other IFD methods. Following initial characterization via dye recovery studies, we demonstrated for the first time that CO2-driven displacement does not inhibit downstream NAATs; NAs isolated direct-from-swab on disc were compatible with both 'gold standard' polymerase chain reaction (PCR) techniques and loop-mediated isothermal amplification (LAMP). The IFD approach described here stands to significantly ease integration of an increased number of sequential on-board processes, including cellular lysis, nucleic acid extraction, amplification, and detection, to greatly lower barriers towards automatable sample-to-answer LoaDs amenable for use on-site operation by non-technical personnel.

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.