Integration of Bioinspired Fibrous Strands with 3D Spheroids for Environmental Hazard Monitoring.

Numerous methods have been introduced to produce 3D cell cultures that can reduce the need for animal experimentation. This study presents a unique 3D culture platform that features bioinspired strands of electrospun nanofibers (BSeNs) and aquatic cell lines to compensate for shortcomings in the current cell spheroid generation techniques. The use of BSeNs in 3D zebrafish liver cell cultures is found to improve liver and reproductive functions through spheroid-based in vitro assays such as whole transcriptome sequencing and reproductive toxicity testing, with optimized properties exhibiting results similar to those obtained for fish embryo acute toxicity (FET, OECD TG 236) following exposure to environmental endocrine-disrupting chemicals (17ß-Estradiol (E2), 4-hydroxytamoxifen (4-HT), and bisphenol compounds (bisphenol A (BPA) and 9,9-Bis(4-hydroxyphenyl)fluorene (BPFL)). These findings indicate that the beneficial effects of bioinspired materials that closely mimic ECM environments can yield efficient zebrafish cells with intrinsic functions and xenobiotic metabolism similar to those of zebrafish embryos. As a closer analog for the in vivo conditions that are associated with exposure to potentially hazardous chemicals, the straightforward culture model introduced in this study shows promise as an alternative tool that can be used to further eco-environmental assessment.

User-Friendly Microfabrication Method for Complex Topological Structure and Three-Dimensional Microchannel with the Application Prospect in Polymerase Chain Reaction (PCR).

One of the most important challenges in the field of microfluidics is the rapid fabrication of microchips with complex topologies. Although the processing method of microfluidic chips has made brilliant achievements in the past 20 years, almost all traditional processing methods still face huge obstacles in the production of complex topologies and three-dimensional microchannel. Nowadays, the main methods of manufacturing microfluidic chips such as numerical control microprocessing, laser ablation, inkjet printing, photolithography, dry etching, and lithography, galvanoformung and abformung (LIGA) technology are not only inapplicable to the complex topological structure and the rapid processing of three-dimensional microfluidic chips but also rely on expensive processing equipment, complex manufacturing process, and low yield. To solve the problems of these traditional processing methods, we propose a low-cost methodology to obtain a microfluidic chip by sewing the chip pipe to the substrate with an embroidery machine as low as $6. Compared with the above-mentioned traditional microprocessing technologies, the new chip processing technology proposed by us does not involve professional microprocessing equipment and professional skills. Therefore, this new chip processing technology can significantly improve the efficiency of microprocessing.

Miniaturized Continuous-Flow Digital PCR for Clinical-Level Serum Sample Based on the 3D Microfluidics and CMOS Imaging Device.

In recent years, the development of polymerase chain reaction (PCR) technology has focused on digital PCR, which depends on the microfluidics. Based on continuous-flow microfluidic technology, this paper designed a miniaturized digital PCR amplification system, and greatly reduced the area required for microdroplet generation and reaction. The core rod. made of polydimethylsiloxane (PDMS), was combined with the Teflon tube to form 3D microfluidics, which requires only one heating source to form the temperature difference required for gene amplification. Only two 34 g needles can form and transmit micro-droplets in a 4-fold tapered Teflon tube, which is the simplest method to generate digital PCR droplets as far as we know, which allows the microdroplet generation device to be free from dependence on expensive chips. A complementary metal oxide semiconductor (CMOS) camera was used as a detection tool to obtain fluorescence video for the entire loop area or a specified loop area. In addition, we developed a homebrew for automatic image acquisition and processing to realize the function of digital PCR. This technique realizes the analysis of clinical serum samples of hepatitis B virus (HBV) and obtained the same results as real-time quantitative PCR. This system has greatly reduced the size and cost of the entire system, while maintaining a stable response.

On-line electroextraction in capillary electrophoresis: Application on the determination of glutamic acid in soy sauces.

We present an on-line, single step coupling between liquid-liquid extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection, which allows an efficient analysis of complex food matrices with high sodium content. The sodium depletion was demonstrated using an aqueous two-phase system. The aqueous two-phase system enables the electrically driven extraction of the target compounds. The sample was prepared in Dextran-rich phase (8% w/v 500 kDa Dextran, DEX). The background electrolyte (acetic acid 5.0 mol/L) contained 6% w/v of 6 kDa PEG. As proof of applicability, we employed the developed method for glutamic acid quantification on soy sauces. The peak area of glutamic acid presents no significant difference (α = 0.05), while the peak area of the sodium presented a reduction of 11.7 ± 0.2 and 19 ± 3% for premium and low-cost soy sauce samples analyzed. The glutamic acid concentration for premium soy sauce sample was 2.7 ± 0.8 and 4.8 ± 0.4 g/L, and for low-cost soy sauce sample, the concentration was 9.9 ± 0.9 g/L, which agreed with those obtained by other analytical techniques.

Long-term observation of Magnetospirillum gryphiswaldense in a microfluidic channel.

We controlled and observed individual magneto-tactic bacteria (Magnetospirillum gryphiswaldense) inside a [Formula: see text]-high microfluidic channel for over 4 h. After a period of constant velocity, the duration of which varied between bacteria, all observed bacteria showed a gradual decrease in their velocity of about [Formula: see text]. After coming to a full stop, different behaviour was observed, ranging from rotation around the centre of mass synchronous with the direction of the external magnetic field, to being completely immobile. Our results suggest that the influence of the high-intensity illumination and the presence of the channel walls are important parameters to consider when performing observations of such long duration.

Study of melatonin-mediated effects on various hepatic inflammatory responses stimulated by IL-6 in a new HepG2-on-a-chip platform.

Hepatocytes exhibit diverse reactions upon stimulation with the interleukin IL-6, mainly in the context of inflammation and energy metabolism. Melatonin has been shown to exert pleiotropic protective actions, such as anti-inflammation and anti-oxidative stress on many cell- and organ-types. The key role of the liver to maintain homeostasis and metabolic regulation prompted us to evaluate the direct modification of IL-6-induced alterations in HepG2 cells in a chip by melatonin. IL-6 administration was followed by the reduced expression and activity of MRP2, a loss of CYP1A activity, and the decline of PXR expression. Other effects were the induction of acute phase responses (reduced albumin production as well as increased CRP and hepcidin expression) and lowered expression of CREB3L3. IL-6 affected also the mitochondrial membrane potential together with elevated mitochondrial superoxide generation, and glycogen deposition was reduced. Melatonin counteracted all observed IL-6-induced alterations except the rise in CRP release and CYP1A activity. Altogether, this new in vitro model can be applied to investigate hepatic inflammatory responses stimulated by IL-6, and these results indicate that hepatocellular inflammatory responses to IL-6 are mitigated by melatonin.

Duplex-imprinted nano well arrays for promising nanoparticle assembly.

A large area nano-duplex-imprint technique is presented in this contribution using natural cicada wings as stamps. The glassy wings of the cicada, which are abundant in nature, exhibit strikingly interesting nanopillar structures over their membrane. This technique, with excellent performance despite the nonplanar surface of the wings, combines both top-down and bottom-up nanofabrication techniques. It transitions micro-nanofabrication from a cleanroom environment to the bench. Two different materials, dicing tape with an acrylic layer and a UV optical adhesive, are used to make replications at the same time, thus achieving duplex imprinting. The promise of a large volume of commercial manufacturing of these nanostructure elements can be envisaged through this contribution to speeding up the fabrication process and achieving a higher throughput. The contact angle of the replicated nanowell arrays before and after oxygen plasma was measured. Gold nanoparticles (50 nm) were used to test how the nanoparticles behaved on the untreated and plasma-treated replica surface. The experiments show that promising nanoparticle self-assembly can be obtained.

Palm-Sized Device for Point-of-Care Ebola Detection.

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics.

Microfluidic Superheating for Peptide Sequence Elucidation.

Herein, we introduce microfluidic superheating as a new method for peptide fragmentation prior to mass spectrometric analysis. The superheating conditions were found to be stable up to 240 °C for more than 30 min without elevated pressure or boiling of the aqueous sample. As proof of principle, we exposed the peptides ACTH1-10 and OVA257-264 to various superheating conditions, causing different degrees of decomposition. Optimized superheating conditions resulted in the entire peptide ladder sequence of the y-ions, allowing the amino acid sequence to be deduced from a single-stage mass spectrum. Thus, obtaining information in the same quality as from tandem mass spectrometry can be achieved by a single superheating step.

Acoustofluidic chemical waveform generator and switch.

Eliciting a cellular response to a changing chemical microenvironment is central to many biological processes including gene expression, cell migration, differentiation, apoptosis, and intercellular signaling. The nature and scope of the response is highly dependent upon the spatiotemporal characteristics of the stimulus. To date, studies that investigate this phenomenon have been limited to digital (or step) chemical stimulation with little control over the temporal counterparts. Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics and microfluidics) approach for generating programmable chemical waveforms that permits continuous modulation of the signal characteristics including the amplitude (i.e., sample concentration), shape, frequency, and duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we show fast switching between multiple distinct stimuli, wherein the waveform of each stimulus is independently controlled. Using our device, we characterized the frequency-dependent activation and internalization of the ß2-adrenergic receptor (ß2-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine. The acoustofluidic-based programmable chemical waveform generation and switching method presented herein is expected to be a powerful tool for the investigation and characterization of the kinetics and other dynamic properties of many biological and biochemical processes.

Terahertz-time domain spectroscopy for the detection of PCR amplified DNA in aqueous solution.

In this work we present a label free quantitative detection method for DNA samples amplified by polymerase chain reaction (PCR) in aqueous medium using terahertz-time domain spectroscopy (THz-TDS) in the frequency range from 0.3 to 1.2 THz. The DNA samples of 133 and 697 base pairs were prepared using PCR. We measured the absorption coefficients of DNA solutions in the concentration range of 0-0.3 ng µl(-1). For both DNA types, the absorption coefficients decreased with increasing DNA concentrations. The average change in absorption coefficients compared to buffer within the frequency range of 0.8-1.0 THz showed a linear behavior. Our results demonstrate that THz-TDS can detect PCR amplified DNA in aqueous solution with a minimum concentration of 0.1 ng µl(-1) and a minimum sample volume of 10 µl.

Scaling and the design of miniaturized chemical-analysis systems.

Micrometre-scale analytical devices are more attractive than their macroscale counterparts for various reasons. For example, they use smaller volumes of reagents and are therefore cheaper, quicker and less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction and detection devices before they are fabricated. Some devices designed using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to arise.

Microfluidic Roadmap for Translational Nanotheranostics.

Nanotheranostic materials (NTMs) shed light on the mechanisms responsible for complex diseases such as cancer because they enable making a diagnosis, monitoring the disease progression, and applying a targeted therapy simultaneously. However, several issues such as the reproducibility and mass production of NTMs hamper their application for clinical practice. To address these issues and facilitate the clinical application of NTMs, microfluidic systems have been increasingly used. This perspective provides a glimpse into the current state-of-art of NTM research, emphasizing the methods currently employed at each development stage of NTMs and the related open problems. This work reviews microfluidic technologies used to develop NTMs, ranging from the fabrication and testing of a single NTM up to their manufacturing on a large scale. Ultimately, a step-by-step vision on the future development of NTMs for clinical practice enabled by microfluidics techniques is provided.

Transcriptomic and physiological analysis of endocrine disrupting chemicals Impacts on 3D Zebrafish liver cell culture system.

In recent decades, extensive efforts have focused on developing in vitro platforms mimicking fish livers to better understand the acute or chronic effects of toxicants on lower aquatic vertebrates. Fish liver cell lines have emerged as a promising culture system for these in vitro platforms because they complement the currently limited in vitro tools that mostly consist of mammalian cell lines and adhere to the 3Rs: replacement, reduction, and refinement of living animal tests. However, monolayer cell lines have lower transcriptional and physiological responses upon exposure to toxic chemicals than freshly isolated primary cells. To overcome this challenge, we utilized a three-dimensional (3D) spheroid-based in vitro platform, in which hepatocyte cells had self-organized into spheroid forms via E-cadherin bonds. This platform exhibited augmented transcriptomic and phenotypic regulation of liver cells in comparison to monolayer cells. We examined the organoid platform using the zebrafish liver (ZFL) cell line as a model system. ZFL cells spontaneously clustered into 3D spheroids with long-term viability by optimizing cell seeding density on a non-adherent substrate. Interestingly, 3D ZFL spheroids treated with estrogenic chemicals were activated to synthesize a higher level of vitellogenin (Vtg) than monolayer cells. Whole-transcriptome sequencing analysis confirmed that 3D ZFL spheroids had greater transcriptional regulation of genes related to reproductive toxicological response and liver functions, such as the urea cycle, estrogen receptors, and vitellogenin, compared to monolayer cells. These results may contribute to the engineering of novel 3D in vitro platforms for screening harmful chemicals and improving understanding of the underlying liver toxicity mechanisms at the molecular and cellular levels.

Targeting extracellular lectins of Pseudomonas aeruginosa with glycomimetic liposomes.

The antimicrobial resistance crisis requires novel approaches for the therapy of infections especially with Gram-negative pathogens. Pseudomonas aeruginosa is defined as priority 1 pathogen by the WHO and thus of particular interest. Its drug resistance is primarily associated with biofilm formation and essential constituents of its extracellular biofilm matrix are the two lectins, LecA and LecB. Here, we report microbial lectin-specific targeted nanovehicles based on liposomes. LecA- and LecB-targeted phospholipids were synthesized and used for the preparation of liposomes. These liposomes with varying surface ligand density were then analyzed for their competitive and direct lectin binding activity. We have further developed a microfluidic device that allowed the optical detection of the targeting process to the bacterial lectins. Our data showed that the targeted liposomes are specifically binding to their respective lectin and remain firmly attached to surfaces containing these lectins. This synthetic and biophysical study provides the basis for future application in targeted antibiotic delivery to overcome antimicrobial resistance.

Fully automatic integrated continuous-flow digital PCR device for absolute DNA quantification.

Existing digital polymerase chain reaction (PCR) devices consist of multiple independent devices, such as a droplet generator, a PCR thermocycler, and a droplet reader, which have the disadvantages of low integration, complex equipment structure, and high operation difficulty. This paper proposes a fully automatic integrated digital PCR device based on continuous-flow digital PCR theory. By simply adding the sample to the entrance of the integrated instrument, a series of procedures required for digital PCR detection can be fully automated, including sample injection, droplet generation, PCR thermal cycling, fluorescence acquisition, and signal analysis. In contrast to traditional techniques, sample testing requires only one integrated device rather than three separate instruments. For full automation, we design complete control and data processing software, which can complete a test by one-step operation. Therefore, the disadvantages of traditional instruments, such as multi-step operation, and hence, potential environmental pollution, are avoided. Moreover, the system can be powered by solar cells and does not require an external power supply. As a proof of concept, the proposed device is used for absolute quantitative detection of the hepatitis B virus in serum samples. The capacity of the system is validated by absolute quantification of three orders of magnitude from 103 to 105 IU/mL. The results have a good linear correlation (0.9986) with those of the traditional quantitative (qPCR), thus confirming the reliability of the instrument. In summary, we believe that our work can promote the development of integrated digital PCR systems.

A digital PCR system based on the thermal cycled chip with multi helix winding capillary.

This paper presents a digital PCR system based on a novel thermal cycled chip, which wraps microchannels on a trapezoidal structure made of polydimethylsiloxane (PDMS) in a multi-helix manner for the first time. It is found that compared to the single helix chip commonly used in previous reports, this kind of novel multi-helix chip can make the surface temperature in the renaturation zone more uniform, and even in the case of rapid fluid flow, it can improve the efficiency of the polymerase chain reaction. What's more, the winding method of multi helix (such as double helix, six helix and eight helix) can obtain better temperature uniformity than the winding of odd helix (such as single helix and three helix). As a proof of concept, the temperature-optimized double-helical chip structure is applied to continuous-flow digital PCR and there is no need to add any surfactant to both the oil phase and reagent. In addition, we successfully analyzed the fluorescence signal of continuous-flow digital PCR by using CMOS camera. Finally, this method is applied for the absolute quantification of the clinical serum sample infected by HBV. The accuracy of the test results has been confirmed by commercial instruments.

Plasmonic heating-based portable digital PCR system.

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s-1 and maximum cooling rate of 8 °C s-1. Target DNA concentrations in the range from 101 ± 1.4 copies per µL to 260 000 ± 20 000 copies per µL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 µm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 µm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per µL to 25 889 ± 737 copies per µL.

Lab-on-a-chip: microfluidics in drug discovery.

Miniaturization can expand the capability of existing bioassays, separation technologies and chemical synthesis techniques. Although a reduction in size to the micrometre scale will usually not change the nature of molecular reactions, laws of scale for surface per volume, molecular diffusion and heat transport enable dramatic increases in throughput. Besides the many microwell-plate- or bead-based methods, microfluidic chips have been widely used to provide small volumes and fluid connections and could eventually outperform conventionally used robotic fluid handling. Moreover, completely novel applications without a macroscopic equivalent have recently been developed. This article reviews current and future applications of microfluidics and highlights the potential of 'lab-on-a-chip' technology for drug discovery.

Differentiation of the human liver progenitor cell line (HepaRG) on a microfluidic-based biochip.

HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.