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.

A Membrane-Modulated Centrifugal Microdevice for Enzyme-Linked Immunosorbent Assay-Based Detection of Illicit and Misused Drugs.

Increased opioid use and misuse have imposed large analytical demands across clinical and forensic sectors. Due to the absence of affordable, accurate, and simple on-site tests (e.g., point of interdiction and bedside), analysis is primarily conducted in centralized laboratories via time-consuming, labor-intensive methods. Many healthcare facilities do not have such analytical capabilities and must send samples to commercial laboratories, increasing turnaround time and care costs, as well as delaying public health warnings regarding the emergence of specific substances. Enzyme-linked immunosorbent assays (ELISAs) are used ubiquitously, despite lengthy workflows that require substantial manual intervention. Faster, reliable analytics are desperately needed to mitigate the mortality and morbidity associated with the current substance use epidemic. We describe one such alternative─a portable centrifugal microfluidic ELISA system that supplants repetitive pipetting with rotationally controlled fluidics. Embedded cellulosic membranes act as microvalves, permitting flow only when centrifugally generated hydraulic pressure exceeds their liquid entry pressure. These features enable stepwise reagent introduction, incubation, and removal simply by tuning rotational frequency. We demonstrate the success of this platform through sensitive, specific colorimetric detection of opiates, a subclass of opioids naturally derived from the opium poppy. Objective image analysis eliminated subjectivity in human color perception and permitted reliable detection of opiates in buffer and artificial urine at the ng/µL range. Opiates were clearly differentiated from other drug classes without interference from common adulterants known to cause false positive results in current colorimetric field tests. Eight samples were simultaneously analyzed in under 1 h, a marked reduction from the traditional multiday timeline. This approach could permit rapid, automatable ELISA-based drug detection outside of traditional laboratories by nontechnical personnel.

Towards an affinity-free, centrifugal microfluidic system for rapid, automated forensic differential extraction.

Biological evidence originating from victims of sexual assault is often comprised of unbalanced cellular mixtures with significantly higher contributions from the victim's genetic material. Enrichment of the forensically-critical sperm fraction (SF) with single-source male DNA relies on differential extraction (DE), a manually-intensive process that is prone to contamination. Due to DNA losses from sequential washing steps, some existing DE methods often fail to generate sufficient sperm cell DNA recovery for perpetrator(s) identification. Here, we propose an enzymatic, 'swab-in' rotationally-driven microfluidic device to achieve complete, self-contained, on-disc automation of the forensic DE workflow. This 'swab-in' approach retains the sample within the microdevice, enabling lysis of sperm cells directly from the evidence cutting to improve sperm cell DNA yield. We demonstrate clear proof-of-concept of a centrifugal platform that provides for timed reagent release, temperature control for sequential enzymatic reactions, and enclosed fluidic fractionation that allows for objective evaluation of the DE process chain with a total processing time of ≤15 min. On-disc extraction of buccal or sperm swabs establishes compatibility of the prototype disc with: 1) an entirely enzymatic extraction method, and 2) distinct downstream analysis modalities, such as the PicoGreen® DNA assay for nucleic acid detection and the polymerase chain reaction (PCR).

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.

Digital postprocessing and image segmentation for objective analysis of colorimetric reactions.

Recently, there has been an explosion of scientific literature describing the use of colorimetry for monitoring the progression or the endpoint result of colorimetric reactions. The availability of inexpensive imaging technology (e.g., scanners, Raspberry Pi, smartphones and other sub-$50 digital cameras) has lowered the barrier to accessing cost-efficient, objective detection methodologies. However, to exploit these imaging devices as low-cost colorimetric detectors, it is paramount that they interface with flexible software that is capable of image segmentation and probing a variety of color spaces (RGB, HSB, Y'UV, L*a*b*, etc.). Development of tailor-made software (e.g., smartphone applications) for advanced image analysis requires complex, custom-written processing algorithms, advanced computer programming knowledge and/or expertise in physics, mathematics, pattern recognition and computer vision and learning. Freeware programs, such as ImageJ, offer an alternative, affordable path to robust image analysis. Here we describe a protocol that uses the ImageJ program to process images of colorimetric experiments. In practice, this protocol consists of three distinct workflow options. This protocol is accessible to uninitiated users with little experience in image processing or color science and does not require fluorescence signals, expensive imaging equipment or custom-written algorithms. We anticipate that total analysis time per region of interest is ~6 min for new users and <3 min for experienced users, although initial color threshold determination might take longer.

A survey of bacteria associated with various life stages of primary colonizers: Lucilia sericata and Phormia regina.

Blow flies are common primary colonizers of carrion, play an important role in the transfer of microbes between environments, and serve as a vector for many human pathogens. While some investigation has begun regarding the bacteria associated with different life stages of blow flies, a well replicated study is currently not available for the majority of blow flies. This study investigated bacteria associated with successive life stages of blow fly species Lucilia sericata and Phormia regina. A total of 38 samples were collected from four true replicates of L. sericata and P. regina. Variable region four (V4) of 16S ribosomal DNA (16S rDNA) was amplified and sequenced on MiSeq FGx sequencing platform using universal 16S rDNA primers and dual-index sequencing strategy. Bacterial communities associated with different life stages of L. sericata and P. regina didn't differ significantly from each other. In both blow fly species, Bacilli (e.g., Lactococcus) and Gammaproteobacteria (e.g., Providencia) constituted >95% of all bacterial classes across all life stages. At the genus level, Vagococcus and Leuconostoc were present at relatively high abundances in L. sericata whereas Yersinia and Proteus were present at comparatively high abundances in P. regina. Overall, information on bacterial structures associated with various life stages of blow flies can help scientists in better understanding or management of vector-borne pathogen dispersal and in increasing the accuracy of microbial evidence based postmortem interval (PMI) prediction models.

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.

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.

Real Time Electronic Feedback for Improved Acoustic Trapping of Micron-Scale Particles.

Acoustic differential extraction has been previously reported as a viable alternative to the repetitive manual pipetting and centrifugation steps for isolating sperm cells from female epithelial cells in sexual assault sample evidence. However, the efficiency of sperm cell isolation can be compromised in samples containing an extremely large number of epithelial cells. When highly concentrated samples are lysed, changes to the physicochemical nature of the medium surrounding the cells impacts the acoustic frequency needed for optimal trapping. Previous work has demonstrated successful, automated adjustment of acoustic frequency to account for changes in temperature and buffer properties in various samples. Here we show that, during acoustic trapping, real-time monitoring of voltage measurements across the piezoelectric transducer correlates with sample-dependent changes in the medium. This is achieved with a wideband peak detector circuit, which identifies the resonant frequency with minimal disruption to the applied voltage. We further demonstrate that immediate, corresponding adjustments to acoustic trapping frequency provides retention of sperm cells from high epithelial cell-containing mock sexual assault samples.