Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

The surface and internal features of pubic hair: A comparative study with those of scalp hair.

Although pubic hair has been a subject of public interest, little is known about its structure or characteristics beyond its curly and coarse appearance. In this study, we investigated the surface and internal features of pubic hair from Korean males and compared them to those of scalp hair from the same donors. Our findings indicate that the cuticle layer of pubic hair has a greater number of scales than that of scalp hair, resulting in a thicker cuticle layer overall. Fourier-transform infrared (FT-IR) spectroscopy analysis showed that the protein in the cortex layer of pubic hair was less affected by exposure to urine or ammonia than the protein in the cortex layer of scalp hair. This suggests that the cuticle layer of pubic hair, which is thicker and composed of more scales, acts as a physical barrier that protects the hair's internal structure. Furthermore, we observed that the secondary and tertiary structures of keratin in the pubic hair cuticle layer are essentially different from those in scalp hair. Based on these findings, we hypothesize that the thickened cuticle layer in pubic hair may have evolved as a defence mechanism against chemical damage from urine, urea and ammonia.

A Self-Regenerating Artificial Cell, that is One Step Closer to Living Cells: Challenges and Perspectives.

Controllable, self-regenerating artificial cells (SRACs) can be a vital advancement in the field of synthetic biology, which seeks to create living cells by recombining various biological molecules in the lab. This represents, more importantly, the first step on a long journey toward creating reproductive cells from rather fragmentary biochemical mimics. However, it is still a difficult task to replicate the complex processes involved in cell regeneration, such as genetic material replication and cell membrane division, in artificially created spaces. This review highlights recent advances in the field of controllable, SRACs and the strategies to achieve the goal of creating such cells. Self-regenerating cells start by replicating DNA and transferring it to a location where proteins can be synthesized. Functional but essential proteins must be synthesized for sustained energy generation and survival needs and function in the same liposomal space. Finally, self-division and repeated cycling lead to autonomous, self-regenerating cells. The pursuit of controllable, SRACs will enable authors to make bold advances in understanding life at the cellular level, ultimately providing an opportunity to use this knowledge to understand the nature of life.

Artificial organelles for sustainable chemical energy conversion and production in artificial cells: Artificial mitochondrion and chloroplasts.

Sustainable energy conversion modules are the main challenges for building complex reaction cascades in artificial cells. Recent advances in biotechnology have enabled this sustainable energy supply, especially the adenosine triphosphate (ATP), by mimicking the organelles, which are the core structures for energy conversion in living cells. Three components are mainly shared by the artificial organelles: the membrane compartment separating the inner and outer parts, membrane proteins for proton translocation, and the molecular rotary machine for ATP synthesis. Depending on the initiation factors, they are further categorized into artificial mitochondrion and artificial chloroplasts, which use chemical nutrients for oxidative phosphorylation and light for photosynthesis, respectively. In this review, we summarize the essential components needed for artificial organelles and then review the recent progress on two different artificial organelles. Recent strategies, purified and identified proteins, and working principles are discussed. With more study on the artificial mitochondrion and artificial chloroplasts, they are expected to be very powerful tools, allowing us to achieve complex cascading reactions in artificial cells, like the ones that happen in real cells.

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite.

The extracellular matrix (ECM) is a network of connective fibers that supports cells living in their surroundings. Native ECM, generated by the secretory products of each tissue's resident cells, has a unique architecture with different protein composition depending on the tissue. Therefore, it is very difficult to artificially design in vivo architecture in tissue engineering. In this study, a hybrid ECM scaffold from the basic structure of fibroblast-derived cellular ECMs is fabricated by adding major ECM components of fibronectin (FN) and collagen (COL I) externally. It is confirmed that while maintaining the basic structure of the native ECM, major protein components can be regulated. Then, decellularization is performed to prepare hybrid ECM scaffolds with various protein compositions and it is demonstrated that a liver-mimicking fibronectin (FN)-rich hybrid ECM promoted successful settling of H4IIE rat hepatoma cells. The authors believe that their method holds promise for the fabrication of scaffolds that provide a tailored cellular microenvironment for specific organs and serve as novel pathways for the replacement or regeneration of specific organ tissues.

Contagious Aggregation: Transmittable Protein Aggregation in Cellular Communities Initiated by Synthetic Cells.

Aggregation of amyloidogenic proteins causing neurodegenerative diseases is an uncontrollable and contagious process that is often associated with lipid membranes in a highly complex physiological environment. Although several approaches using natural cells and membrane models have been reported, systematic investigations focusing on the association with the membranes are highly challenging, mostly because of the lack of proper molecular tools. Here, we report a new supramolecular approach using a synthetic cell system capable of controlling the initiation of protein aggregation and mimicking various conditions of lipid membranes, thereby enabling systematic investigations of membrane-dependent effects on protein aggregation by visualization. Extending this strategy through concurrent use of synthetic cells and natural cells, we demonstrate the potential of this approach for systematic and in-depth studies on interrogating inter- and intracellularly transmittable protein aggregation. Thus, this new approach offers opportunities for gaining insights into the pathological implications of contagious protein aggregation associated with membranes for neurotoxicity.

Pumpless three-dimensional photo paper-based microfluidic analytical device for automatic detection of thioredoxin-1 using enzyme-linked immunosorbent assay.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-µPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-µPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-µPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-µPAD, it can detect 0-200 ng/mL of Trx-1, and the prepared 3D-µPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-µPAD, and comparing results to ELISA, it can be confirmed that the 3D-µPAD is a good tool for cancer diagnosis.

An Extracellular Matrix-Liposome Composite, a Novel Extracellular Matrix Delivery System for Accelerated Tissue Regeneration.

The unfolded states of fibronectin (FN) subsequently induce the formation of an extracellular matrix (ECM) fibrillar network, which is necessary to generate new substitutive tissues. Here, the authors demonstrate that negatively charged small unilamellar vesicles (SUVs) qualify as candidates for FN delivery due to their remarkable effects on the autonomous binding and unfolding of FN, which leads to increased tissue regeneration. In vitro experiments revealed that the FN-SUV complex remarkably increased the attachment, differentiation, and migration of fibroblasts. The potential utilization of this complex in vivo to treat inflammatory colon diseases is also described based on results obtained for ameliorated conditions in rats with ulcerative colitis (UC) that had been treated with the FN-SUV complex. Their findings provide a new ECM-delivery platform for ECM-based therapeutic applications and suggest that properly designed SUVs may be an unprecedented FN-delivery system that is highly effective in treating UC and inflammatory bowel diseases.

Controlled metabolic cascades for protein synthesis in an artificial cell.

In recent years, researchers have been pursuing a method to design and to construct life forms from scratch - in other words, to create artificial cells. In many studies, artificial cellular membranes have been successfully fabricated, allowing the research field to grow by leaps and bounds. Moreover, in addition to lipid bilayer membranes, proteins are essential factors required to construct any cellular metabolic reaction; for that reason, different cell-free expression systems under various conditions to achieve the goal of controlling the synthetic cascades of proteins in a confined area have been reported. Thus, in this review, we will discuss recent issues and strategies, enabling to control protein synthesis cascades that are being used, particularly in research on artificial cells.

Discovery of a microbial rhodopsin that is the most stable in extreme environments.

Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology.

Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA).

This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

Scalable Superior Chemical Sensing Performance of Stretchable Ionotronic Skin via a π-Hole Receptor Effect.

Skin-attachable gas sensors provide a next-generation wearable platform for real-time protection of human health by monitoring environmental and physiological chemicals. However, the creation of skin-like wearable gas sensors, possessing high sensitivity, selectivity, stability, and scalability (4S) simultaneously, has been a big challenge. Here, an ionotronic gas-sensing sticker (IGS) is demonstrated, implemented with free-standing polymer electrolyte (ionic thermoplastic polyurethane, i-TPU) as a sensing channel and inkjet-printed stretchable carbon nanotube electrodes, which enables the IGS to exhibit high sensitivity, selectivity, stability (against mechanical stress, humidity, and temperature), and scalable fabrication, simultaneously. The IGS demonstrates reliable sensing capability against nitrogen dioxide molecules under not only harsh mechanical stress (cyclic bending with the radius of curvature of 1 mm and cyclic straining at 50%), but also environmental conditions (thermal aging from -45 to 125 °C for 1000 cycles and humidity aging for 24 h at 85% relative humidity). Further, through systematic experiments and theoretical calculations, a π-hole receptor mechanism is proposed, which can effectively elucidate the origin of the high sensitivity (up to parts per billion level) and selectivity of the ionotronic sensing system. Consequently, this work provides a guideline for the design of ionotronic materials for the achievement of high-performance and skin-attachable gas-sensor platforms.

A Simple Route of Printing Explosive Crystalized Micro-Patterns by Using Direct Ink Writing.

The production of energetic crystalized micro-patterns by using one-step printing has become a recent trend in energetic materials engineering. We report a direct ink writing (DIW) approach in which micro-scale energetic composites composed of 1,3,5-trinitro-1,3,5-triazinane (RDX) crystals in selected ink formulations of a cellulose acetate butyrate (CAB) matrix are produced based on a direct phase transformation from organic, solvent-based, all-liquid ink. Using the formulated RDX ink and the DIW method, we printed crystalized RDX micro-patterns of various sizes and shapes on silicon wafers. The crystalized RDX micro-patterns contained single crystals on pristine Si wafers while the micro-patterns containing dendrite crystals were produced on UV-ozone (UVO)-treated Si wafers. The printing method and the formulated all-liquid ink make up a simple route for designing and printing energetic micro-patterns for micro-electromechanical systems.

An Accelerated Wound-Healing Surgical Suture Engineered with an Extracellular Matrix.

A suture is a ubiquitous medical device to hold wounded tissues together and support the healing process after surgery. Surgical sutures, having incomplete biocompatibility, often cause unwanted infections or serious secondary trauma to soft or fragile tissue. In this research, UV/ozone (UVO) irradiation or polystyrene sulfonate acid (PSS) dip-coating is used to achieve a fibronectin (FN)-coated absorbable suture system, in which the negatively charged moieties produced on the suture cause fibronectin to change from a soluble plasma form into a fibrous form, mimicking the actions of cellular fibronectin upon binding. The fibrous fibronectin coated on the suture can be exploited as an engineered interface to improve cellular migration and adhesion in the region around the wounded tissue while preventing the binding of infectious bacteria, thereby facilitating wound healing. Furthermore, the FN-coated suture is found to be associated with a lower friction between the suture and the wounded tissue, thus minimizing the occurrence of secondary wounds during surgery. It is believed that this surface modification can be universally applied to most kinds of sutures currently in use, implying that it may be a novel way to develop a highly effective and safer suture system for clinical applications.

A bioinspired and hierarchically structured shape-memory material.

Shape-memory polymeric materials lack long-range molecular order that enables more controlled and efficient actuation mechanisms. Here, we develop a hierarchical structured keratin-based system that has long-range molecular order and shape-memory properties in response to hydration. We explore the metastable reconfiguration of the keratin secondary structure, the transition from α-helix to ß-sheet, as an actuation mechanism to design a high-strength shape-memory material that is biocompatible and processable through fibre spinning and three-dimensional (3D) printing. We extract keratin protofibrils from animal hair and subject them to shear stress to induce their self-organization into a nematic phase, which recapitulates the native hierarchical organization of the protein. This self-assembly process can be tuned to create materials with desired anisotropic structuring and responsiveness. Our combination of bottom-up assembly and top-down manufacturing allows for the scalable fabrication of strong and hierarchically structured shape-memory fibres and 3D-printed scaffolds with potential applications in bioengineering and smart textiles.

Recent Advances in Microfluidic Paper-Based Analytical Devices toward High-Throughput Screening.

Microfluidic paper-based analytical devices (µPADs) have become promising tools offering various analytical applications for chemical and biological assays at the point-of-care (POC). Compared to traditional microfluidic devices, µPADs offer notable advantages; they are cost-effective, easily fabricated, disposable, and portable. Because of our better understanding and advanced engineering of µPADs, multistep assays, high detection sensitivity, and rapid result readout have become possible, and recently developed µPADs have gained extensive interest in parallel analyses to detect biomarkers of interest. In this review, we focus on recent developments in order to achieve µPADs with high-throughput capability. We discuss existing fabrication techniques and designs, and we introduce and discuss current detection methods and their applications to multiplexed detection assays in relation to clinical diagnosis, drug analysis and screening, environmental monitoring, and food and beverage quality control. A summary with future perspectives for µPADs is also presented.

Formulation of Sugar/Hydrogel Inks for Rapid Thermal Response 4D Architectures with Sugar-derived Macropores.

Programmed, reshaping hydrogel architectures were fabricated from sugar/hydrogel inks via a three-dimensional printing method involving a stimuli-responsive polymer. We developed a new hydrogel ink composed of monomers (acrylamide [AAm]) and N-isopropylacrylamide [NIPAAm]), and sugar (mixture of glucose and sucrose) as a pore-generator, enabling to improve printability by increasing the ink's viscoelastic properties and induce the formation of macropores in the hydrogel architectures. This study demonstrated that creating macropores in such architectures enables rapid responses to stimuli that can facilitate four-dimensional printing. We printed bilayer structures from monomer inks to which we had added sugar, and we exposed them to processes that cross-linked the monomers and leached out the sugar to create macropores. In comparison with a conventional poly(N-isopropylacrylamide) hydrogel, the macroporous hydrogels prepared using polymerization in the presence of a high concentration of sugar showed higher swelling ratios and exhibited much faster response rates to temperature changes. We used rheometry and scanning electron microscopy to characterize the properties of these inks and hydrogels. The results suggest that this method may provide a readily available route to the rapid design and fabrication of shape-morphing hydrogel architectures with potential application in soft robotics, hydrogel actuators, and tissue engineering.