Nucleic Acid Detection with Ion Concentration Polarization Microfluidic Chip for Reduced Cycle Numbers of Polymerase Chain Reaction.

Nucleic acid detection is widely used in disease diagnosis, food safety, environmental monitoring and many other research fields. The continuous development of rapid and sensitive new methods to detective nucleic acid is very important for practical application. In this study, we developed a rapid nucleic-acid detection method using polymerase chain reaction (PCR) combined with electrokinetic preconcentration based on ion concentration polarization (ICP). Using a Nafion film, the proposed ICP microfluidic chip is utilized to enrich the nucleic acid molecules amplified by PCR thermal cycles. To demonstrate the capability of the microfluidic device and the hybrid nucleic-acid detection method, we present an animal-derived component detection experiment for meat product identification applications. With the reduced cycle numbers of 24 cycles, the detection can be completed in about 35 min. The experimental results show that this work can provide a microfluidic device and straightforward method for rapid detection of nucleic acids with reduced cycle numbers.

A Cost-Effective Nucleic Acid Detection System Using a Portable Microscopic Device.

A fluorescence microscope is one of the most important tools for biomedical research and laboratory diagnosis. However, its high cost and bulky size hinder the application of laboratory microscopes in space-limited and low-resource applications. Here, in this work, we proposed a portable and cost-effective fluorescence microscope. Assembled from a set of 3D print components and a webcam, it consists of a three-degree-of-freedom sliding platform and a microscopic imaging system. The microscope is capable of bright-field and fluorescence imaging with micron-level resolution. The resolution and field of view of the microscope were evaluated. Compared with a laboratory-grade inverted fluorescence microscope, the portable microscope shows satisfactory performance, both in the bright-field and fluorescence mode. From the configurations of local resources, the microscope costs around USD 100 to assemble. To demonstrate the capability of the portable fluorescence microscope, we proposed a quantitative polymerase chain reaction experiment for meat product authenticating applications. The portable and low-cost microscope platform demonstrates the benefits in space-constrained environments and shows high potential in telemedicine, point-of-care testing, and more.

Microfluidics Temperature Compensating and Monitoring Based on Liquid Metal Heat Transfer.

Microfluidic devices offer excellent heat transfer, enabling the biochemical reactions to be more efficient. However, the precision of temperature sensing and control of microfluids is limited by the size effect. Here in this work, the relationship between the microfluids and the glass substrate of a typical microfluidic device is investigated. With an intelligent structure design and liquid metal, we demonstrated that a millimeter-scale industrial temperature sensor could be utilized for temperature sensing of micro-scale fluids. We proposed a heat transfer model based on this design, where the local correlations between the macro-scale temperature sensor and the micro-scale fluids were investigated. As a demonstration, a set of temperature-sensitive nucleic acid amplification tests were taken to show the precision of temperature control for micro-scale reagents. Comparations of theoretical and experimental data further verify the effectiveness of our heat transfer model. With the presented compensation approach, the slight fluorescent intensity changes caused by isothermal amplification polymerase chain reaction (PCR) temperature could be distinguished. For instance, the probability distribution plots of fluorescent intensity are significant from each other, even if the amplification temperature has a difference of 1 °C. Thus, this method may serve as a universal approach for micro-macro interface sensing and is helpful beyond microfluidic applications.

Easy-to-Operate Co-Flow Step Emulsification Device for High-Throughput Three-Dimensional Cell Culture.

Cell culture plays an essential role in tissue engineering and high-throughput drug screening. Compared with two-dimensional (2D) in vitro culture, three-dimensional (3D) in vitro culture can mimic cells in vivo more accurately, including complex cellular organizations, heterogeneity, and cell-extracellular matrix (ECM) interactions. This article presents a droplet-based microfluidic chip that integrates cell distribution, 3D in vitro cell culture, and in situ cell monitoring in a single device. Using the microfluidic "co-flow step emulsification" approach, we have successfully prepared close-packed droplet arrays with an ultra-high-volume fraction (72%) which can prevent cells from adhering to the chip surface so as to achieve a 3D cell culture and make scalable and high-throughput cell culture possible. The proposed device could produce droplets from 55.29 ± 1.52 to 95.64 ± 3.35 µm, enabling the diverse encapsulation of cells of different sizes and quantities. Furthermore, the cost for each microfluidic CFSE chip is approximately USD 3, making it a low-cost approach for 3D cell culture. The proposed device is successfully applied in the 3D culture of saccharomyces cerevisiae cells with an occurrence rate for proliferation of 80.34 ± 3.77%. With low-cost, easy-to-operate, high-throughput, and miniaturization characteristics, the proposed device meets the requirements for 3D in vitro cell culture and is expected to be applied in biological fields such as drug toxicology and pharmacokinetics.

Easy-to-Operate Co-flow Step Emulsification Device for Droplet Digital Polymerase Chain Reaction.

Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/µL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.

NIR light-responsive bacteria with live bio-glue coatings for precise colonization in the gut.

Recombinant bacterial colonization plays an indispensable role in disease prevention, alleviation, and treatment. Successful application mainly depends on whether bacteria can efficiently spatiotemporally colonize the host gut. However, a primary limitation of existing methods is the lack of precise spatiotemporal regulation, resulting in uncontrolled methods that are less effective. Herein, we design upconversion microgels (UCMs) to convert near-infrared light (NIR) into blue light to activate recombinant light-responsive bacteria (Lresb) in vivo, where autocrine "functional cellular glues" made of adhesive proteins assist Lresb inefficiently colonizing the gut. The programmable engineering platform is further developed for the controlled and effective colonization of Escherichia coli Nissle 1917 (EcN) in the gut. The colonizing bacteria effectively alleviate DSS-induced colitis in mice. We anticipate that this approach could facilitate the clinical application of engineered microbial therapeutics to accurately and effectively regulate host health.

Rapid Microfluidic Mixing Method Based on Droplet Rotation Due to PDMS Deformation.

Droplet-based micromixers have shown great prospects in chemical synthesis, pharmacology, biologics, and diagnostics. When compared with the active method, passive micromixer is widely used because it relies on the droplet movement in the microchannel without extra energy, which is more concise and easier to operate. Here we present a droplet rotation-based microfluidic mixer that allows rapid mixing within individual droplets efficiently. PDMS deformation is used to construct subsidence on the roof of the microchannel, which can deviate the trajectory of droplets. Thus, the droplet shows a rotation behavior due to the non-uniform distribution of the flow field, which can introduce turbulence and induce cross-flow enhancing 3D mixing inside the droplet, achieving rapid and homogenous fluid mixing. In order to evaluate the performance of the droplet rotation-based microfluidic mixer, droplets with highly viscous fluid (60% w/w PEGDA solution) were generated, half of which was seeded with fluorescent dye for imaging. Mixing efficiency was quantified using the mixing index (MI), which shows as high as 92% mixing index was achieved within 12 mm traveling. Here in this work, it has been demonstrated that the microfluidic mixing method based on the droplet rotation has shown the advantages of low-cost, easy to operate, and high mixing efficiency. It is expected to find wide applications in the field of pharmaceutics, chemical synthesis, and biologics.

Pre-Degassed Microfluidic Chamber-Based Digital PCR Device for Meat Authentication Applications.

Droplet digital polymerase chain reaction (ddPCR) suffers from the need for specific equipment and skilled personnel; thus, we here present a chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a pre-degassed microfluidic device that consists of a pilot channel and micro chamber arrays. Digitalized PCR reagents are introduced into micro chambers, and thermocycles are taken to perform a DNA amplification process. Then, fluorescence images of a micro chamber array are read out and analyzed to obtain the total number of positive chambers. Thereby, the copy numbers of target DNA are calculated for quantitative detections. As a validation, this device is evaluated by the application of meat authentication. We performed dPCR tests using DNA templates extracted from a pure mutton DNA template with different dilutions. Then, the dPCR chip was used to identify the meat authentication using mutton-chicken mixtures with different mass ratios, showing its performance in real biotechnical applications.

A Microfluidic Concentration Gradient Maker with Tunable Concentration Profiles by Changing Feed Flow Rate Ratios.

Microfluidic chips-in which chemical or biological fluid samples are mixed into linear or nonlinear concentration distribution profiles-have generated enormous enthusiasm of their ability to develop patterns for drug release and their potential toxicology applications. These microfluidic devices have untapped potential for varying concentration patterns by the use of one single device or by easy-to-operate procedures. To address this challenge, we developed a soft-lithography-fabricated microfluidic platform that enabled one single device to be used as a concentration maker, which could generate linear, bell-type, or even S-type concentration profiles by tuning the feed flow rate ratios of each independent inlet. Here, we present an FFRR (feed flow rate ratio) adjustment approach to generate tens of types of concentration gradient profiles with one single device. To demonstrate the advantages of this approach, we used a Christmas-tree-like microfluidic chip as the demo. Its performance was analyzed using numerical simulation models and experimental investigations, and it showed an excellent time response (~10 s). With on-demand flow rate ratios, the FFRR microfluidic device could be used for many lab-on-a-chip applications where flexible concentration profiles are required for analysis.

Pressure-Driven Micro-Casting for Electrode Fabrication and Its Applications in Wear Grain Detections.

The microelectrode is an essential and vital part in microsensors that are largely used in industrial, chemical, and biological applications. To obtain desired microelectrodes in great quality, it is also of great necessity and significance to develop a robust method to fabricate the microelectrode pattern. This work developed a four-terminal differential microelectrode that aims at recognizing microparticles in fluids. This microelectrode pair consisted of a high height-width ratio microelectrode array fabricated using a pre-designed microelectrode pattern (a micro-scale channel) and melted liquid metal. The surface treatment of microelectrodes was also investigated to reveal its impacts on the continuality of melting metal and the quality of the fabricated microelectrode patterns. To evaluate the performance of micro-casting fabricated electrodes, a microfluidic device was packaged using a microelectrode layer and a flow layer. Then impedance cytometer experiments were performed using sample fluids with polymer particles in two different sizes in diameter (5 µm and 10 µm). In addition, engine oil was tested on the microelectrodes as complex samples. The number of abrasive particles in the engine oil can be collected from the developed microfluidic device for further analysis.

Cloning of the Lycopene ß-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene ß cyclase (ß-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a ß-LCY gene from Nicotiana tabacum, designated as Ntß-LCY1, was cloned and functionally characterized. Robust expression of Ntß-LCY1 was found in leaves, and Ntß-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntß-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntß-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntß-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Nicorandil inhibits oxidative stress and amyloid-ß precursor protein processing in SH-SY5Y cells overexpressing APPsw.

It has been demonstrated that ATP-sensitive potassium (KATP) channel activation has neuroprotective effects against neuronal damage induced by hypoxia, ischemia or metabolism stress. This study investigated the multiply protective effects of KATP channel opener nicorandil against neurotoxicity in SH-SY5Y cells transiently transfected with Swedish mutant APP (APPsw) and also the potential involvement of PI3K/Akt/GSK-3ß pathway. Cells were treated with nicorandil (1 mM) for 24 h with and without glibenclamide (10 µM), a KATP channel inhibitor. Then the cells were collected for Hoechst33342, biochemical assays, real-time PCR, western blot and ELISA assay. Our results showed that nicorandil reduced apoptosis and decreased oxidative stress. Moreover, nicorandil down regulated APP695 mRNA and APP695 protein expression, also reduced Aß1-42 levels in the medium. In addition, nicorandil increased the protein levels of p-Akt and p-GSK-3ß by PI3K activation. Applying a PI3K inhibitor, LY294002 blocked the protection. These findings suggest nicorandil to be a potential therapeutic agent to treat Alzheimer's disease (AD).

Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of ß-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of ß-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Thermal decomposition pathways of hydroxylamine: theoretical investigation on the initial steps.

Hydroxylamine (NH(2)OH) is an unstable compound at room temperature, and it has been involved in two tragic industrial incidents. Although experimental studies have been carried out to study the thermal stability of hydroxylamine, the detailed decomposition mechanism is still in debate. In this work, several density functional and ab initio methods were used in conjunction with several basis sets to investigate the initial thermal decomposition steps of hydroxylamine, including both unimolecular and bimolecular reaction pathways. The theoretical investigation shows that simple bond dissociations and unimolecular reactions are unlikely to occur. The energetically favorable initial step of decomposition pathways was determined as a bimolecular isomerization of hydroxylamine into ammonia oxide with an activation barrier of approximately 25 kcal/mol at the MPW1K level of theory. Because hydroxylamine is available only in aqueous solutions, solvent effects on the initial decomposition pathways were also studied using water cluster methods and the polarizable continuum model (PCM). In water, the activation barrier of the bimolecular isomerization reaction decreases to approximately 16 kcal/mol. The results indicate that the bimolecular isomerization pathway of hydroxylamine is more favorable in aqueous solutions. However, the bimolecular nature of this reaction means that more dilute aqueous solution will be more stable.

Hydroxylamine nitrate self-catalytic kinetics study with adiabatic calorimetry.

Hydroxylamine nitrate (HAN) is an important member of the hydroxylamine compound family with applications that include equipment decontamination in the nuclear industry and aqueous or solid propellants. Due to its instability and autocatalytic behavior, HAN has been involved in several incidents at the Hanford and Savannah River Site (SRS) [Technical Report on Hydroxylamine Nitrate, US Department of Energy, 1998]. Much research has been conducted on HAN in different areas, such as combustion mechanism, decomposition mechanism, and runaway behavior. However, the autocatalytic decomposition behavior of HAN at runaway stage has not been fully addressed due to its highly exothermic and rapid decomposition behavior. This work is focused on extracting HAN autocatalytic kinetics and analyzing HAN critical behavior from adiabatic calorimetry measurements. A lumped autocatalytic kinetic model for HAN and associated model parameters are determined. Also the storage and handling critical conditions of diluted HAN solution without metal presence are quantified.

Layer of protection analysis for reactive chemical risk assessment.

Reactive chemical hazards have been a significant concern for the chemical process industries (CPI). Without sufficient control and mitigation of chemical reaction hazards, reactive incidents have led to severe consequences, such as release of flammable and toxic materials, fires and explosions, and threats to human lives, properties, and the environment. Consequence of reactive hazards can be well understood through calorimetric testing and computational techniques. However, risks of incidents caused by reactive chemicals have not been well addressed due partly to sparse failure frequency data. In this paper, the semi-quantitative layer of protection analysis (LOPA) approach is used to estimate reactive chemical risk, and the probabilities or frequencies of failure scenarios are addressed. Using LOPA, reactive risks can be evaluated with respect to predefined criteria, and the effectiveness of risk reduction measures can be assessed. The hydroxylamine (HA) production system is employed as a case study to demonstrate the application of LOPA to reactive chemical risk assessment.

Thermal decomposition hazard evaluation of hydroxylamine nitrate.

Hydroxylamine nitrate (HAN) is an important member of the hydroxylamine family and it is a liquid propellant when combined with alkylammonium nitrate fuel in an aqueous solution. Low concentrations of HAN are used primarily in the nuclear industry as a reductant in nuclear material processing and for decontamination of equipment. Also, HAN has been involved in several incidents because of its instability and autocatalytic decomposition behavior. This paper presents calorimetric measurement for the thermal decomposition of 24 mass% HAN/water. Gas phase enthalpy of formation of HAN is calculated using both semi-empirical methods with MOPAC and high-level quantum chemical methods of Gaussian 03. CHETAH is used to estimate the energy release potential of HAN. A Reactive System Screening Tool (RSST) and an Automatic Pressure Tracking Adiabatic Calorimeter (APTAC) are used to characterize thermal decomposition of HAN and to provide guidance about safe conditions for handling and storing of HAN.