Sample-to-answer sensing technologies for nucleic acid preparation and detection in the field.

Efficient sample preparation and accurate disease diagnosis under field conditions are of great importance for the early intervention of diseases in humans, animals, and plants. However, in-field preparation of high-quality nucleic acids from various specimens for downstream analyses, such as amplification and sequencing, is challenging. Thus, developing and adapting sample lysis and nucleic acid extraction protocols suitable for portable formats have drawn significant attention. Similarly, various nucleic acid amplification techniques and detection methods have also been explored. Combining these functions in an integrated platform has resulted in emergent sample-to-answer sensing systems that allow effective disease detection and analyses outside a laboratory. Such devices have a vast potential to improve healthcare in resource-limited settings, low-cost and distributed surveillance of diseases in food and agriculture industries, environmental monitoring, and defense against biological warfare and terrorism. This paper reviews recent advances in portable sample preparation technologies and facile detection methods that have been / or could be adopted into novel sample-to-answer devices. In addition, recent developments and challenges of commercial kits and devices targeting on-site diagnosis of various plant diseases are discussed.

Emerging Technologies for Monitoring Plant Health in Vivo.

In the coming decades, increasing agricultural productivity is all-important. As the global population is growing rapidly and putting increased demand on food supply, poor soil quality, drought, flooding, increasing temperatures, and novel plant diseases are negatively impacting yields worldwide. One method to increase yields is plant health monitoring and rapid detection of disease, nutrient deficiencies, or drought. Monitoring plant health will allow for precise application of agrichemicals, fertilizers, and water in order to maximize yields. In vivo plant sensors are an emerging technology with the potential to increase agricultural productivity. In this mini-review, we discuss three major approaches of in vivo sensors for plant health monitoring, including genetic engineering, imaging and spectroscopy, and electrical.

Laser-etched grooves for rapid fluid delivery for a paper-based chemiresistive biosensor.

Paper-based microfluidic devices are an attractive option for developing low-cost, point-of-care diagnostic tools. To incorporate more complex assays into paper, these devices must become more sophisticated, through the sequential delivery of different liquids or reagents without user intervention. Many flow control strategies focus on slowing the fluid down. However, this can lead to increased assay times and sample loss due to evaporation. We report the use of a CO2 laser to create etched grooves on paper to accelerate wicking speeds in paper-based microfluidic devices. We explored different laser settings to determine the optimal configuration. Our findings showed that simply cutting a slit into the paper created the fastest wicking channels. The slit acted as a macro capillary, allowing fluid to bypass the paper and speed it up. Further studies determined an ideal groove pitch of 0.75 mm (spacing in between grooves) for a paper channel. Additional experiments documented how sealing grooved channels with different adhesives can influence wicking. Overall, sealing the channels with tape made them wick faster. However, sealing methods such as lamination had a negative effect on wicking. Laser-etched grooves were successfully used to design a fluid-handling architecture for a chemiresistive paper-based biosensor. The grooves facilitated rapid, sequential delivery of sample and wash buffer. Human serum albumin spiked in phosphate buffer, artificial urine, and artificial saliva was successfully detected at as low as 15 pM. Etching grooves in paper is a simple process that requires no additional materials or chemicals, allowing single-step fabrication of paper-based microfluidic channels.

Recent developments in flow modeling and fluid control for paper-based microfluidic biosensors.

Over the last 10 years, researchers have shown that paper is a promising substrate for affordable biosensors. The field of paper-microfluidics has evolved rapidly in that time, with simple colorimetric assays giving way to more complex electrochemical devices that can handle multiple samples at a given time. As paper devices become more complex, the ability to precisely control different fluids simultaneously becomes a challenge. Specifically, automated flow control is a necessary attribute to make paper-based devices more useable in resource-limited settings. Flow control strategies on paper are typically developed experimentally through trial-and-error, with little focus on theory. This is because flow behavior in paper is not well understood and sometimes difficult to predict precisely. Additionally, popular theoretical models are too simplistic, making them unsuitable for complex device designs and application conditions. A better understanding of flow theory would allow devices conceived straight from theoretical models. This could save time and resources by reducing experimental work. In this review, we provide an overview of different theoretical models used to characterize imbibition in paper substrates and document the latest flow control strategies that have been applied to automated fluid control on paper. Additionally, we look at current efforts to commercialize paper-based devices along with challenges facing this industry.

An origami electrical biosensor for multiplexed analyte detection in body fluids.

We developed an affordable, highly sensitive, and specific paper-based microfluidic platform for fast multiplexed detections of important biomarkers in various body fluids, including urine, saliva, serum, and whole blood. The sensor array consisted of five individual sensing channels with various functionalities that only required a micro liter-sized sample, which was equally split into aliquots by the built-in paper microfluidics. We achieved the individual functionalizations of various bioreceptors by employing the use of wax barriers and 'paper bridges' in an easy and low-cost manner. Pyrene carboxylic acid-modified single-walled carbon nanotubes (PCA/SWNTs) were deposited by quantitative inkjet printing with an optimal 3-dimensional semiconductor density on a paper substrate. Multiple antibodies were immobilized onto the SWNTs surface for highly sensitive and specific field-effect transistor (FET)/chemiresistor (CR) biosensors. We explored the optimal sensing conditions for the paper-based CR biosensor to achieve high sensitivities and specificities towards the target biomarker proteins (human serum albumin (HSA) and human immunoglobulin G (HIgG)) and achieved an ultralow detectable concentration of HSA and HIgG at 1.5 pM. Besides, origami folding was employed to simplify the fabrication process further. The sensing platform described in this work was cost-effective, semi-automated, and user-friendly. It demonstrated the capability of having multiple sensing functions in one paper-based microfluidic sensing platform. It envisioned the potential of a point-of-care device with full-analysis for practical diagnostics in an ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) fashion for a quick test of targets of interest.

Modifying Wicking Speeds in Paper-Based Microfluidic Devices by Laser-Etching.

Paper-based microfluidic devices are an attractive platform for developing low-cost, point-of-care diagnostic tools. As paper-based devices' detection chemistries become more complex, more complicated devices are required, often entailing the sequential delivery of different liquids or reagents to reaction zones. Most research into flow control has been focused on introducing delays. However, delaying the flow can be problematic due to increased evaporation leading to sample loss. We report the use of a CO2 laser to uniformly etch the surface of the paper to modify wicking speeds in paper-based microfluidic devices. This technique can produce both wicking speed increases of up to 1.1× faster and decreases of up to 0.9× slower. Wicking speeds can be further enhanced by etching both sides of the paper, resulting in wicking 1.3× faster than unetched channels. Channels with lengthwise laser-etched grooves were also compared to uniformly etched channels, with the most heavily grooved channels wicking 1.9× faster than the fastest double-sided etched channels. Furthermore, sealing both sides of the channel in packing tape results in the most heavily etched channels, single-sided, double-sided, and grooved, wicking over 13× faster than unetched channels. By selectively etching individual channels, different combinations of sequential fluid delivery can be obtained without altering any channel geometry. Laser etching is a simple process that can be integrated into the patterning of the device and requires no additional materials or chemicals, enabling greater flow control for paper-based microfluidic devices.

Distance and Microsphere Aggregation-Based DNA Detection in a Paper-Based Microfluidic Device.

In paper-based microfluidics, the simplest devices are colorimetric, giving qualitative results. However, getting quantitative data can be quite a bit more difficult. Distance-based devices provide a user-friendly means of obtaining quantitative data without the need for any additional equipment, simply by using an included ruler or calibrated markings. This article details the development of a quantitative DNA detection device that utilizes the aggregation of polystyrene microspheres to affect the distance that microspheres wick through filter paper. The microspheres are conjugated to single-stranded DNA (ssDNA) oligomers that are partially complementary to a target strand and, in the presence of the target strand, form a three-strand complex, resulting in the formation of aggregates. The higher the concentration of the target strand, the larger the aggregate, and the shorter the distance wicked by the microspheres. This behavior was investigated across a wide range of target concentrations and under different incubation times to understand aggregate formation. The device was then used to successfully detect a target strand spiked in extracted plant DNA.

Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery.

Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular.

A paper-based chemiresistive biosensor employing single-walled carbon nanotubes for low-cost, point-of-care detection.

Paper-based biosensors are promising for low-cost diagnostics. However, its widespread use has been hampered due to a lack of sensitive detection methods that can be easily implemented on paper substrates. On the other hand, single-walled carbon nanotubes (SWNTs) -based chemiresistive biosensors are gaining popularity as label-free, highly sensitive biosensors. However, traditional SWNT-based chemiresistors need to be more affordable for use in resource-limited settings. In this study, we report fabrication, optimization and analytical characterization of a chemiresistive biosensor on paper for label-free immunosensing. We synthesized a water-based ink using pyrene carboxylic acid (PCA) through non-covalent π-π stacking interaction between PCA and SWNTs. The PCA/SWNTs ink concentration can reach ~4 mg mL-1 and was stable at room temperature for one month. We introduced a combination of wax printing and vacuum filtration to fabricate the hydrophilic channels and the well-defined PCA/SWNTs ink deposition on paper in a facile manner requiring no additional masks or stencils. Specific antibodies were then functionalized on the PCA/SWNTs. Quantitative and selective detection of human serum albumin (HSA) is demonstrated with a limit of detection (LOD) of 1 pM. This low LOD is attributed to the porous structure of the paper surface, which can accommodate more SWNTs. Furthermore, the hydroxyl group-containing cellulose fibers help connect the SWNTs into an electrical network. The paper-based chemiresistive biosensor proposed here is easy to fabricate, and designed for rapid, sensitive and selective detection of HSA. This work provides a potential platform for automated, disposable paper-based biosensors with multiplexed detection capability and microfluidic controls.

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro.

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows across a large spatiotemporal range. However, its implementation typically requires the use of expensive and specialized instrumentation, which limits its broader utility. Moreover, within the field of bioengineering, in vitro flow visualization studies are also often further limited by the high cost of commercially sourced tissue phantoms that recapitulate desired anatomical structures, particularly for those that span the mesoscale regime (i.e., submillimeter to millimeter length scales). Herein, we present a simplified experimental protocol developed to address these limitations, the key elements of which include 1) a relatively low-cost method for fabricating mesoscale tissue phantoms using 3-D printing and silicone casting, and 2) an open-source image analysis and processing framework that reduces the demand upon the instrumentation for measuring mesoscale flows (i.e., velocities up to tens of millimeters/second). Collectively, this lowers the barrier to entry for nonexperts, by leveraging resources already at the disposal of many bioengineering researchers. We demonstratethe applicability of this protocol within the context of neurovascular flow characterization; however, it is expected to be relevant to a broader range of mesoscale applications in bioengineering and beyond.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc.

Polydiacetylene-Coated Sensor Strip for Immunochromatic Detection of Xylella fastidiosa subsp. fastidiosa.

This study presents a sensor strip for user-friendly, naked-eye detection of Xylella fasitdiosa, the bacterial causal agent of Pierce's disease in grapevine. This sensor uses anti- X. fastidiosa antibodies conjugated to a polydiacetylene layer on a polyvinylidene fluoride strip to generate specific color transitions and discriminate levels of the pathogen. The detection limit of the sensor is 0.8 × 108 cells/mL, which is similar to bacterial load in grapevine 18 days following bacterial inoculation. This sensor enables equipment-free detection that is highly desirable for in-field diagnostic tools in resource-limited settings.

Polydiacetylene-coated polyvinylidene fluoride strip aptasensor for colorimetric detection of zinc(II).

We report a new polydiacetylene (PDA) sensor strip for simple visual detection of zinc ions in aqueous solution. The specificity of this sensor comes from Zn2+ DNA aptamer probes conjugated onto PDA. Effects of aptamer length and structure on the sensitivity of PDA's color transition were first investigated. PDA conjugated with the optimal aptamer sequence was then coated onto a strip of polyvinylidene fluoride membrane and photopolymerized by UV exposure. The newly developed sensor successfully exhibited a blue-to-red chromatic change in a semi-quantitative manner in response to zinc ions. No discernable change was observed in solutions containing other common ions. Advantages of this sensor include its ease of fabrication, high specificity, and equipment-free detection, all of which are desirable for in-field applications and use in resource-limited settings.

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

In Planta Microsphere-Based Lateral Flow Leaf Biosensor in Maize.

Low-cost and quick detection of biotic stresses is critically important for protection of staple food crops such as maize in smallholder farms in developing countries, where access to improved seed varieties, fertilizers, and pesticides is limited due to financial and geographical reasons. Here, we report a new lateral flow detection technology directly integrated in a maize leaf, in which microspheres conjugated with analyte-specific capture antibodies are non-invasively injected. The antibody-conjugated microspheres capture and detect an analyte in a concentration-specific manner. In this study, we optimized microsphere size for effective infiltration and immobilization in the leaf, and further demonstrated detection of a fluorescent mock biomarker, fluorescein, in a live maize plant. This in planta lateral flow biosensor is the first of its kind and is expected to provide a low-cost and user-friendly detection method for biotic stresses in the field.

Patterned adhesive enables construction of nonplanar three-dimensional paper microfluidic circuits.

This article discusses the fabrication of planar and nonplanar 3D paper microfluidic circuits through the use of patterned spray adhesive application and origami techniques. The individual paper layers are held together via semi-permanent adhesive bonds without the need for external clamps. Semi-permanent bonds accommodate the repeated folding and unfolding required by complex origami device structures and allow the device to be unfolded post-use to view internally displayed results. Combinations of adhesive patterns and fluid channel widths were identified that did not prevent the fluid from traveling between layers and through the entire circuit. Further, this method was extended to nonplanar 3D paper microfluidic circuits, demonstrated via multi-fluid wicking within a modified origami peacock. Such nonplanar 3D paper microfluidic circuits are expected to offer an entirely new platform for exploring new designs and functions of paper analytical devices.

Pathophysiology and treatment of patients with globus sensation--from the viewpoint of esophageal motility dysfunction.

"Globus sensation" is often described as the sensation of a lump in the throat associated with dry swallowing or the need for dry swallowing, which disappears completely during eating or drinking and for which no organic cause can be established. Due to the uncertain etiology of "globus sensation", it remains difficult to establish standard treatment strategies for affected patients. Lately most attention has been focused on gastroesophageal reflux disease and several reports have indicated that there is a close relationship between esophageal acid reflux and globus sensation. Nowadays, empirical therapy with a high dose of a proton pump inhibitor (PPI) is considered to be indicated for patients with globus sensation, after excluding organic diseases such as pharyngeal cancer, Zenker's diverticulum, or thyroid enlargement. If patients are nonresponsive to PPI therapy, evaluation of esophageal motility should be done. In our recent study, 47.9% had abnormal esophageal motility, with the most common esophageal motility abnormality being an ineffective esophageal motility in PPI-resistant patients with globus sensation. This suggests that prokinetics alone or adding prokinetics to PPI should be the treatment to be considered, although few studies have investigated the efficacy of prokinetics in the treatment of patients with globus sensation. If patients without any esophageal motility dysfunctions are nonresponsive to PPI therapy, either cognitive-behavioral therapy, anti-depressants, or gabapentin could be helpful, although further well-designed, randomized controlled large-scale studies will be necessary to determine the effectiveness of each treatment strategy on patients with globus sensation.

Engineered micromechanical cues affecting human pluripotent stem cell regulations and fate.

The survival, growth, self-renewal, and differentiation of human pluripotent stem cells (hPSCs) are influenced by their microenvironment, or so-called "niche," consisting of particular chemical and physical cues. Previous studies on mesenchymal stem cells and other stem cells have collectively uncovered the importance of physical cues and have begun to shed light on how stem cells sense and process such cues. In an attempt to support similar progress in mechanobiology of hPSCs, we review mechanosensory machinery, which plays an important role in cell-extracellular matrix interactions, cell-cell interactions, and subsequent intracellular responses. In addition, we review recent studies on the mechanobiology of hPSCs, in which engineered micromechanical environments were used to investigate effects of specific physical cues. Identifying key physical cues and understanding their mechanism will ultimately help in harnessing the full potential of hPSCs for clinical applications.