Bio-Inspired Microreactors Continuously Synthesize Glucose Precursor from CO2 with an Energy Conversion Efficiency 3.3 Times of Rice.

Excessive CO2 and food shortage are two grand challenges of human society. Directly converting CO2 into food materials can simultaneously alleviate both, like what green crops do in nature. Nevertheless, natural photosynthesis has a limited energy efficiency due to low activity and specificity of key enzyme D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). To enhance the efficiency, many prior studies focused on engineering the enzymes, but this study chooses to learn from the nature to design more efficient reactors. This work is original in mimicking the stacked structure of thylakoids in chloroplasts to immobilize RuBisCO in a microreactor using the layer-by-layer strategy, obtaining the continuous conversion of CO2 into glucose precursor at 1.9 nmol min-1 with enhanced activity (1.5 times), stability (≈8 times), and reusability (96% after 10 reuses) relative to the free RuBisCO. The microreactors are further scaled out from one to six in parallel and achieve the production at 15.8 nmol min-1 with an energy conversion efficiency of 3.3 times of rice, showing better performance of this artificial synthesis than NPS in terms of energy conversion efficiency. The exploration of the potential of mass production would benefit both food supply and carbon neutralization.

All-perfluoropolymer, nonlinear stability-assisted monolithic surface combines topology-specific superwettability with ultradurability.

Developing versatile and robust surfaces that mimic the skins of living beings to regulate air/liquid/solid matter is critical for many bioinspired applications. Despite notable achievements, such as in the case of developing robust superhydrophobic surfaces, it remains elusive to realize simultaneously topology-specific superwettability and multipronged durability owing to their inherent tradeoff and the lack of a scalable fabrication method. Here, we present a largely unexplored strategy of preparing an all-perfluoropolymer (Teflon), nonlinear stability-assisted monolithic surface for efficient regulating matters. The key to achieving topology-specific superwettability and multilevel durability is the geometric-material mechanics design coupling superwettability stability and mechanical strength. The versatility of the surface is evidenced by its manufacturing feasibility, multiple-use modes (coating, membrane, and adhesive tape), long-term air trapping in 9-m-deep water, low-fouling droplet transportation, and self-cleaning of nanodirt. We also demonstrate its multilevel durability, including strong substrate adhesion, mechanical robustness, and chemical stability, all of which are needed for real-world applications.

Microfluidic synthesis as a new route to produce novel functional materials.

By geometrically constraining fluids into the sub-millimeter scale, microfluidics offers a physical environment largely different from the macroscopic world, as a result of the significantly enhanced surface effects. This environment is characterized by laminar flow and inertial particle behavior, short diffusion distance, and largely enhanced heat exchange. The recent two decades have witnessed the rapid advances of microfluidic technologies in various fields such as biotechnology; analytical science; and diagnostics; as well as physical, chemical, and biological research. On the other hand, one additional field is still emerging. With the advances in nanomaterial and soft matter research, there have been some reports of the advantages discovered during attempts to synthesize these materials on microfluidic chips. As the formation of nanomaterials and soft matters is sensitive to the environment where the building blocks are fed, the unique physical environment of microfluidics and the effectiveness in coupling with other force fields open up a lot of possibilities to form new products as compared to conventional bulk synthesis. This Perspective summarizes the recent progress in producing novel functional materials using microfluidics, such as generating particles with narrow and controlled size distribution, structured hybrid materials, and particles with new structures, completing reactions with a quicker rate and new reaction routes and enabling more effective and efficient control on reactions. Finally, the trend of future development in this field is also discussed.

The Application of Microfluidic Technologies in Aptamer Selection.

Aptamers are sequences of single-strand oligonucleotides (DNA or RNA) with potential binding capability to specific target molecules, which are increasingly used as agents for analysis, diagnosis, and medical treatment. Aptamers are generated by a selection method named systematic evolution of ligands by exponential enrichment (SELEX). Numerous SELEX methods have been developed for aptamer selections. However, the conventional SELEX methods still suffer from high labor intensity, low operation efficiency, and low success rate. Thus, the applications of aptamer with desired properties are limited. With their advantages of low cost, high speed, and upgraded extent of automation, microfluidic technologies have become promising tools for rapid and high throughput aptamer selection. This paper reviews current progresses of such microfluidic systems for aptamer selection. Comparisons of selection performances with discussions on principles, structure, operations, as well as advantages and limitations of various microfluidic-based aptamer selection methods are provided.

"Barcode" cell sensor microfluidic system: Rapid and sample-to-answer antimicrobial susceptibility testing applicable in resource-limited conditions.

Many rapid antimicrobial susceptibility testing (AST) methods have been proposed to contain clinical antimicrobial resistance (AMR) and preserve the effectiveness of remaining antimicrobials. However, far fewer methods have been proposed to test AMR in resource-limited conditions, such as for frequent safety screenings of water/food/public facilities, urgent surveys of massive samples during a pandemic, or AMR tests in low-income countries. Rapid AST methods realized thus far have a variety of drawbacks when used for such surveys, e.g., high cost and the requirement of expensive instruments such as microscopy. A more reasonable strategy would be to screen samples via onsite testing first, and then send any sample suspected to contain AMR bacteria for advanced testing. Accordingly, a cost-efficient AST is demanded, which can rapidly process a large number of samples without using expensive equipment. To this end, current work demonstrates a novel "barcode" cell sensor based on an adaptive linear filter array as a fully automatic and microscope-free method for counting very small volumes of cells (~1.00 × 104 cells without pre-incubation), wherein suspended cells concentrate into microbars with length proportional to the number of cells. We combined this sensor with an on-chip culture approach we had demonstrated for rapid and automated drug exposure and realized a low-cost and resource-independent platform for portable AST, from which results can be obtained simply through a cell phone. This method has a much shorter turnaround time (2-3 h) than that of standard methods (16-24 h). Thanks to its microscopy-free analysis, affordability, portability, high throughput, and user-friendliness, our "barcode" AST system has the potential to fulfill the various demands of AST when advanced facilities are not available, making it a promising new tool in the fight against AMR.

Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis.

Mimicking the cellular environment, metal-organic frameworks (MOFs) are promising for encapsulating enzymes for general applications in environments often unfavorable for native enzymes. Markedly different from previous researches based on bulk solution synthesis, here, we report the synthesis of enzyme-embedded MOFs in a microfluidic laminar flow. The continuously changed concentrations of MOF precursors in the gradient mixing on-chip resulted in structural defects in products. This defect-generating phenomenon enables multimodal pore size distribution in MOFs and therefore allows improved access of substrates to encapsulated enzymes while maintaining the protection to the enzymes. Thus, the as-produced enzyme-MOF composites showed much higher (~one order of magnitude) biological activity than those from conventional bulk solution synthesis. This work suggests that while microfluidic flow synthesis is currently underexplored, it is a promising strategy in producing highly active enzyme-MOF composites.

Crack engineering for the construction of arbitrary hierarchical architectures.

Three-dimensional hierarchical morphologies widely exist in natural and biomimetic materials, which impart preferential functions including liquid and mass transport, energy conversion, and signal transmission for various applications. While notable progress has been made in the design and manufacturing of various hierarchical materials, the state-of-the-art approaches suffer from limited materials selection, high costs, as well as low processing throughput. Herein, by harnessing the configurable elastic crack engineering-controlled formation and configuration of cracks in elastic materials-an effect normally avoided in various industrial processes, we report the development of a facile and powerful technique that enables the faithful transfer of arbitrary hierarchical structures with broad material compatibility and structural and functional integrity. Our work paves the way for the cost-effective, large-scale production of a variety of flexible, inexpensive, and transparent 3D hierarchical and biomimetic materials.

Reliable and reusable whole polypropylene plastic microfluidic devices for a rapid, low-cost antimicrobial susceptibility test.

Using an antimicrobial susceptibility test (AST) as an example, this work demonstrates a practical method to fabricate microfluidic chips entirely from polypropylene (PP) and the benefits for potential commercial use. Primarily caused by the misuse and abuse of antibiotics, antimicrobial resistance (AMR) is a major threat to modern medicine. The AST is a promising technique to help with the optimal use of antibiotics for reducing AMR. However, current phenotypic ASTs suffer from long turnaround time, while genotypic ASTs suffer from low reliability, and both are unaffordable for routine use. New microfluidics based AST methods are rapid but still unreliable as well as costly due to the PDMS chip material. Herein, we demonstrate a convenient method to fabricate whole PP microfluidic chips with high resolution and fidelity. Unlike PDMS chips, the whole PP chips showed better reliability due to their inertness; they are solvent-compatible and can be conveniently reused and recycled, which largely decreases the cost, and are environmentally friendly. We specially designed 3D chambers that allow for quick cell loading without valving/liquid exchange; this new hydrodynamic design satisfies the shear stress requirement for on-chip bacterial culture, which, compared to reported designs for similar purposes, allows for a simpler, more rapid, and high-throughput operation. Our system allows for reliable tracking of individual cells and acquisition of AST results within 1-3 hours, which is among the group of fastest phenotypic methods. The PP chips are more reliable and affordable than PDMS chips, providing a practical solution to improve current culture-based AST and benefiting the fight against AMR through helping doctors prescribe effective, narrow-spectrum antibiotics; they will also be broadly useful for other applications wherein a reliable, solvent-resistant, anti-fouling, and affordable microfluidic chip is needed.

Microfluidic technologies for vasculature biomimicry.

Microfluidic technology has been extensively employed in biology and medicine since the field emerged in the 1990s. By utilizing microfluidic approaches, a variety of vascular system-related structures and functions have been mimicked on in vitro platforms. Herein, we begin by introducing microfluidic circulatory devices for the study of two-dimensional (2D) endothelial cells culture. Next, we focus on recent progress on on-chip mimicry of native vasculature, specifically generation of complex three-dimensional (3D) structures within cell-laden hydrogels using microfluidics and self-assembly-based methods. The utilization of microfluidic technology will facilitate the construction of progressively biomimetic in vitro models that have great potential in complementing existing animal models. We envision such platforms to be utilized in a wide range of applications involving vascular systems, including microphysiological studies, drug screening, and disease modeling.

A portable oligonucleotide-based microfluidic device for the detection of VEGF165 in a three-step suspended-droplet mode.

Vascular endothelial growth factor (VEGF165), an important glycosylated protein from the VEGF family, is a type of signal protein highly associated with the development and progression of cancers. In this work, we designed a G-quadruplex-based aptasensing platform for the sensitive and selective detection of VEGF165 in aqueous solution and red blood cell solution. A long-lived phosphorescence iridium(iii) complex (1) with promising photophysical properties and a large Stokes shift was chosen as a selective G-quadruplex probe. The platform could achieve a limit of detection (LOD) down to the picomolar level using a conventional fluorometer. Furthermore, we successfully applied the platform to a three-step suspended droplet (SD)-based microfluidic device for the monitoring of VEGF165. In contrast to the channel-based and digital microfluidic chips, SD-based chips allow easy introduction of liquid samples, valve-free manipulation of multiple reaction steps and flexible volume range. Importantly, polypropylene (PP), a hydrophobic and thermally stable material, was chosen as a substrate to fabricate the chip for the SD-based microfluidic device. The PP-based chip allows the combination of superhydrophobic force, gravity and surface tension for effective driving of the suspended droplet throughout the channel without reverse migration. After assembling all the major components, including a UV lamp, a rotatable chip holder, a filter and a camera into the portable device, we successfully demonstrated the applicability of the device to detect VEGF165 in aqueous solution with a LOD of 0.33 nM at a signal-to-noise ratio (S/N) of 3 and a linear range of 1-100 nM.

A suspending-droplet mode paper-based microfluidic platform for low-cost, rapid, and convenient detection of lead(II) ions in liquid solution.

A paper-based microfluidic device based on unconventional principle was developed and used to detect lead ions through a two-step process including heated incubation and subsequent mixing. The device was made by generating a superhydrophobic pattern, which defines channel and reservoir barriers, on a water-impermeable paper substrate, followed by loading and drying the reagents in the defined reservoirs. Different from the conventional paper-based devices that are made of water-permeable paper, the as-prepared device holds water drops in discrete reservoirs, and the water drops will not move unless the device is titled along the direction of the predefined channels. In this way, the liquid samples applied onto the device are handled as individual drops and could be stored, transported, and mixed on demand. Different from the conventional paper-based devices that use capillary force to drive liquid, our new device uses wetting and gravity as driving force. We name this operation principle suspending-droplet mode paper-based device (SD-µPAD). The use of a Teflon contact-printing stamp makes the production of such devices rapid, cost efficient, and mass productive. Utilizing a G-quadruplex-based luminescence switch-on assay, we demonstrated rapid, convenient, highly sensitive, and low cost detection of lead(II) ions in water samples, using a custom made battery-powered portable device, and a smart phone as the detector.

Microfluidics for Combating Antimicrobial Resistance.

The ever-growing threat of antimicrobial resistance (AMR) demands immediate countermeasures. With its novelty and enabling features including downscaled analysis, precisely controlled local environment, and enhanced speed, accuracy, and cost-efficiency, microfluidics has demonstrated potential in several key areas, including furthering our understanding of bacteria, developing better susceptibility testing tools, and overcoming obstacles in discovery and research of new antibiotics. While ample research results in the field of microfluidics are available, their transformation into practical application is still lagging far behind. We believe that the challenge of AMR will give microfluidics a much-needed opportunity to leap from research papers to true productivity, and gain wider acceptance as a mature technology.

A Multiplexed, Gradient-Based, Full-Hydrogel Microfluidic Platform for Rapid, High-Throughput Antimicrobial Susceptibility Testing.

Antimicrobial resistance has become an immediate threat to modern healthcare systems as it continues to spread across the globe. As development of novel antibiotics stalls, preserving the effectiveness of existing agents has become a priority. One of the major driving forces behind antimicrobial resistance is the misuse and overuse of antibiotics, often a result of data on the susceptibility of pathogens not being obtained in a convenient and timely manner, a need that conventional antimicrobial susceptibility testing struggles to meet. Here, a hydrogel microfluidic platform is reported for antimicrobial susceptibility testing purposes, capable of handling real samples and yielding results within 2.5 h of culture. By using a multiplayer design with channels crossing overhead of each other, multiple experiments, either one- or two-dimensional, can be staged on the same device. Bacteria grown on the surface of the hydrogel can be easily visualized with standard Gram staining after being transferred onto a glass slide. Coupled with software-based image analysis, the system can yield a variety of useful information on bacterial susceptibility and the effects of drugs, such as minimum inhibitory concentration and morphological changes in bacteria, either individually or in combination. Compared to conventional testing methods, this system requires less labor, reagents, and equipment to operate, and has significantly higher speed and efficiency.

The application of a G-quadruplex based assay with an iridium(iii) complex to arsenic ion detection and its utilization in a microfluidic chip.

In this work, the iridium(iii) complex 1 was synthesized and employed in constructing an assay which is based on a G-quadruplex for detecting arsenic ions in aqueous solution. The assay achieved a detection limit of 7.6 nM (ca. 0.57 µg L-1) and showed high selectivity towards arsenic ions over other metal ions. Additionally, the assay could function in natural water and a simple microfluidic chip was used to investigate the potential of this platform for real-time detection.

A one-step strategy for ultra-fast and low-cost mass production of plastic membrane microfluidic chips.

An ultra-fast, extremely cost-effective, and environmentally friendly method was developed for fabricating flexible microfluidic chips with plastic membranes. With this method, we could fabricate plastic microfluidic chips rapidly (within 12 seconds per piece) at an extremely low cost (less than $0.02 per piece). We used a heated perfluoropolymer perfluoroalkoxy (often called Teflon PFA) solid stamp to press a pile of two pieces of plastic membranes, low density polyethylene (LDPE) and polyethylene terephthalate (PET) coated with an ethylene-vinyl acetate copolymer (EVA). During the short period of contact with the heated PFA stamp, the pressed area of the membranes permanently bonded, while the LDPE membrane spontaneously rose up at the area not pressed, forming microchannels automatically. These two regions were clearly distinguishable even at the micrometer scale so we were able to fabricate microchannels with widths down to 50 microns. This method combines the two steps in the conventional strategy for microchannel fabrication, generating microchannels and sealing channels, into a single step. The production is a green process without using any solvent or generating any waste. Also, the chips showed good resistance against the absorption of Rhodamine 6G, oligonucleotides, and green fluorescent protein (GFP). We demonstrated some typical microfluidic manipulations with the flexible plastic membrane chips, including droplet formation, on-chip capillary electrophoresis, and peristaltic pumping for quantitative injection of samples and reagents. In addition, we demonstrated convenient on-chip detection of lead ions in water samples by a peristaltic-pumping design, as an example of the application of the plastic membrane chips in a resource-limited environment. Due to the high speed and low cost of the fabrication process, this single-step method will facilitate the mass production of microfluidic chips and commercialization of microfluidic technologies.

Freestanding 3-D microvascular networks made of alginate hydrogel as a universal tool to create microchannels inside hydrogels.

The diffusion of molecules such as nutrients and oxygen through densely packed cells is impeded by blockage and consumption by cells, resulting in a limited depth of penetration. This has been a major hurdle to a bulk (3-D) culture. Great efforts have been made to develop methods for generating branched microchannels inside hydrogels to support mass exchange inside a bulk culture. These previous attempts faced a common obstacle: researchers tried to fabricate microchannels with gels already loaded with cells, but the fabrication procedures are often harmful to the embedded cells. Herein, we present a universal strategy to create microchannels in different types of hydrogels, which effectively avoids cell damage. This strategy is based on a freestanding alginate 3-D microvascular network prepared by in-situ generation of copper ions from a sacrificial copper template. This alginate network could be used as implants to create microchannels inside different types of hydrogels. This approach effectively addresses the issue of cell damage during microfabrication and made it possible to create microchannels inside different types of gels. The microvascular network produced with this method is (1) strong enough to allow handling, (2) biocompatible to allow cell culturing, and (3) appropriately permeable to allow diffusion of small molecules, while sufficiently dense to prevent blocking of channels when embedded in different types of gels. In addition, composite microtubules could be prepared by simply pre-loading other materials, e.g., particles and large biomolecules, in the hydrogel. Compared with other potential strategies to fabricate freestanding gel channel networks, our method is more rapid, low-cost and scalable due to parallel processing using an industrially mass-producible template. We demonstrated the use of such vascular networks in creating microchannels in different hydrogels and composite gels, as well as with a cell culture in a nutrition gradient based on microfluidic diffusion. In this way, the freestanding hydrogel vascular network we produced is a universal functional unit that can be embedded in different types of hydrogel; users will be able to adopt this strategy to achieve vascular mass exchange in the bulk culture without changing their current protocol. The method is readily implementable to applications in vascular tissue regeneration, drug discovery, 3-D culture, etc.

Cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility.

Antimicrobial resistance (AMR) is a rapidly increasing threat to the effective treatment of infectious diseases worldwide. The two major remedies include: (1) using narrow-spectrum antibiotics based on rapid diagnosis; and (2) developing new antibiotics. A key part of both remedies is the antimicrobial susceptibility test (AST). However, the current standard ASTs that monitor colony formation are costly and time-consuming and the new strategies proposed are not yet practical to be implemented. Herein, we report a strategy to fabricate whole-hydrogel microfluidic chips using alginate-doped agar. This agar-based microfabrication makes it possible to prepare inexpensive hydrogel devices, and allows a seamless link between microfluidics and conventional agar-based cell culture. Different from common microfluidic systems, in our system the cells are cultured on top of the device, similar to normal agar plate culture; on the other hand, the microfluidic channels inside the hydrogel allow precise generation of linear gradient of drugs, thus giving a better performance than the conventional disk diffusion method. Cells in this system are not exposed to any shear flow, which allows the reliable tracking of individual cells and AST results to be obtained within 2-3 hours. Furthermore, our system could test the synergistic effect of drugs through two-dimensional gradient generation. Finally, the platform could be directly implemented to new drug discovery and other applications wherein a fast, cost-efficient method for studying the response of microorganisms upon drug administration is desirable.

Correction: LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.ppat.1004376.].

G-quadruplex-based logic gates for HgII and AgI ions employing a luminescent iridium(iii) complex and extension of metal-mediated base pairs by polymerase.

We report herein the synthesis of a series of cyclometallated iridium(iii) complexes as luminescent G-quadruplex-selective probes, which were used to construct AND, OR and INHIBIT logic gates for the detection of HgII and AgI ions. To our knowledge, this is the first time that the C-AgI-T mismatched base pair has been used for the construction of luminescent assays or logic gates.

LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.