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.

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.

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.

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.

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.

The Effect of Cholesterol on Membrane Binding and Self-Assembly of Collagen Fibrils.

Collagen is a skeleton of native extracellular matrix (ECM) that is known to provide mechanical and structural stability. In an attempt to develop a new connective cellular model with the surrounding ECM without further experimental complications, such as the reconstitution of ECM receptors, we designed the experiments and discovered that the fibrillogenesis of membrane-bound collagen is not spontaneous as it is in the form of free collagen in bulk solution. The confocal microscopic results suggest that cholesterol is a crucial component that facilitates the fibril formation on the membrane surface. In situ X-ray and neutron reflectivity on Langmuir monolayer and solid-supported lipid bilayer models, respectively, reveal two features of cholesterol effects on the collagen fibril formation. Mainly, cholesterol increases the lateral lipid headgroup separation on the membrane surface, which promotes the association degree of collagen monomers. It also enhances the elastic modulus of the membrane to impede membrane filtration by the collagen assemblies.

3D paper-based microfluidic device: a novel dual-detection platform of bisphenol A.

A novel platform of microfluidic paper-based analytical devices (µPADs) for dual detection of bisphenol A (BPA), a model analyte, was fabricated using an electronic digital plotter to create the stacked layer of µPADs and generate the lateral-flow channel without using an external pump. Two detection techniques, including electrochemical detection and laser desorption ionization mass spectrometric detection (LDI-MS), were used complementarily to improve the precision in the detection of BPA. The fluid sample was delivered to both detection zones by the capillary action, automatically generated from the fabricated microfluidic device. For an electrochemical sensor, two ballpoint pens filled with silver nanoparticles (AgNPs) and multiwall carbon nanotube (MWCNT) ink were used to print onto the paper with a contact printing method using a digital plotter. To further improve the sensitivity, zinc oxide (ZnO) was used to modify both electrochemical and LDI-MS detection zones. For BPA detection, high electrocatalytic properties and strong UV absorption of ZnO promote the electron transfer in the electrochemical sensor and ionization efficiency in LDI-MS with low interferences compared with a conventional organic matrix. Under optimal conditions, this platform showed a dual detection capability for BPA with a detection limit of 0.35 µM for electrochemical detection and with an ultralow detection limit of 0.01 pM for LDI-MS. This novel platform might be very useful for trace analyses requiring high precision detection of various analytes.

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.

Cross-Linked Polymersomes with Reversible Deformability and Oxygen Transportability.

Polymersomes are of interest as nanocarriers due to their physical and chemical robustness, which arises from the macromolecular nature of their block copolymer components. However, the physical robustness of polymersomes impairs transmembrane diffusion and responsiveness to mechanical forces. Polymer nanocarriers that can reversibly deform under stress while maintaining structural integrity and transmembrane diffusivity are desired for development of gas transport vehicles. Here, we report polymersomes composed of amphiphilic block copolymers containing polydimethylsiloxane with side-chain pendant vinyl groups. A reversibly deformable polymersome compartmentalizing membrane was obtained by cross-linkage of PEG- b-poly(dimethyl- r-methylvinyl)silane in a self-assembled bilayer via photoradical generation in aqueous media. The covalently cross-linked polymersomes exhibited superior physical robustness compared to unlinked polymersomes while maintaining deformability under stress. Transmembrane oxygen diffusion was confirmed when lumen-encapsulated Zn-porphyrin generated singlet O2 under irradiation, and the anthracene-9,10-dipropionic acid O2 quencher was consumed. Polymersome-encapsulated hemoglobin bound oxygen reversibly, indicating the polymersomes could be used as O2 carriers that reversibly deform without sacrificing structural integrity or oxygen transportability.

Fabrication of Wettability-Patterned Surface for Cellular Micropatterning Using Step-Wise Ion Beam Processing.

In this study, the fabrication of a wettability patterned surface for cellular micropatterning was investigated using step-wise ion beam processing. A perfluorinated poly(ethylene-co-propylene) (FEP) film was first irradiated using accelerated Xe+ ions with 100 keV of energy at the low current density of 1 µA/cm² over the entire surface. Second, its confined regions were irradiated at the higher current density of 15 µA/cm² at various ion fluences through the pattern mask to generate patterns with big differences in wettability. From the analytic results, it was clearly verified that the step-wise irradiation induced effective chemical and morphological changes on the FEP surface, resulting in the successful formation of well-defined micropatterns with relatively hydrophilic and superhydrophobic surfaces. Moreover, the results of in-vitro cell culture showed well-resolved formation of 200 µm cell micropatterns on the wettability patterned FEP surface due to the individual effects of the relatively hydrophilic and superhydrophobic properties on the cell adhesiveness and proliferation.

Preparation of Conductive Carbon Films from Novolac Resins by Ion Beam Irradiation and Carbonization.

In this study, thin carbon films with good electrical properties were prepared using commercial novolac resins by ion beam irradiation and carbonization. Novolac films were irradiated with ion beams and then carbonized under inert atmosphere. Based on the FTIR and UV results, the novolac resins were found to be crosslinked by ion beam irradiation without any additives. The Raman and XRD results indicate that carbon films with pseudo-graphitic structures were formed by carbonization of the ion beam irradiated novolac films. The sheet resistance of the prepared carbon films decreased to 1.35 × 102 Ω/ with an increasing fluence. The prepared carbon films showed a good electrical conductivity of ∼2.34 × 102 S/cm.

Electric Heating Performance of Pyrolyzed Photoresist Films Prepared by Proton Irradiation and Pyrolysis.

In this study, pyrolyzed photoresist films (PPFs) were prepared using commercial SU8 photoresist by proton irradiation and pyrolysis. SU8 thin films were irradiated with high-energy proton ions and then pyrolyzed in a tube furnace at 1000 °C under inert atmosphere. The carbonization yield of the PPFs increased with an increasing fluence due to the formation of more crosslinked network structures at a higher fluence. The electrical resistance decreased with an increasing fluence due to the higher remaining thickness and carbonization yield at a higher fluence. Therefore, the PPFs prepared at 1 × 1016 ions/cm2 showed the maximum temperature of 150 °C at 20 V and a high electric power efficiency of 1.57 mW/°C.

Effects of Silicone Oil on Electrowetting to Actuate a Digital Microfluidic Drop on Paper.

The effects of an immiscible, lubricating polydimethylsiloxane fluid, referred to as silicone oil, on the static deformation and on the dynamic motion of a water drop on paper induced by electrowetting were investigated. The deformation of a drop on a hydrophobic film of amorphous fluoropolymers top-coated with less hydrophobic silicone oil was much more predictable, reversible and reproducible than on the uncoated surface. In the dynamic tribological experiment for a sliding drop along an inclined surface, a significant decrease in the friction coefficient, with an unexpected dependency of the contact area, was observed. Based on the curve fitting analysis, the shear stress and the net friction force were estimated quantitatively. Because of the tribological effect and the reduced shear friction force of the oil film, the static and the dynamic electrowetting states of the water drop were enhanced.

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on a Lipid Membrane.

The bioinspired design of ligands for nanoparticle coating with remarkable precision in controlling anisotropic connectivity and with universal binding efficiency to the membrane has made a great impact on nanoparticle self-assembly. We utilize the HIV-1-derived trans-activator of transcription peptide (TAT), a member of the cell-penetrating peptides, as a soft shell coating on gold nanoparticles (GNPs) and characterize TAT pepide-mediated binding behaviors of GNPs on the lipid membrane. Whereas the peptides enable GNPs to firmly attach to the membrane, the binding structures are driven by two electrostatic forces: the interparticle peptide repulsion and the peptide-membrane attraction. Although transmission electron microscopy images showed that the densities of membrane-embedded GNPs were almost equal, X-ray reflectivity revealed a significant difference in binding structures of GNPs along the surface normal upon the increase of charge densities (ϕ) of the membrane. In particular, GNPs were densely suspended at ϕ = 70% while they adopted an additional well-defined layer underneath the membrane at ϕ = 100%, in addition to a translocation of the initially bound particles into the membrane. The observed behaviors of GNPs manifest a 3D to 2D transformation of the self-assembled structures from the diffused state to the closely packed state with the increase in the charge density of the membrane. The present study also provides insights on the binding mechanisms of the cell-penetrating peptide-coated nanoparticles to the lipid membranes, which is a common theme of delivery systems in pharmaceutical research.

Effects of cardiolipin on membrane morphology: a Langmuir monolayer study.

Cardiolipin (CL) is a complex phospholipid that is specifically found in mitochondria. Owing to the association of the CL levels with mitochondrial physiopathology such as in Parkinson's disease, we study the molecular effect of CL on membrane organization using model Langmuir monolayer, fluorescence microscopy, and x-ray reflectivity. We find that the liquid-expanded phase in membranes increases with increasing CL concentration, indicating an increase in the elasticity of the mixed membrane. The Gibbs excess free energy of mixing indicates that the binary monolayer composed of CL and DPPC is most thermodynamically stable at ΦCL = 10 mol%, and the stability is enhanced when the surface pressure is increased. Additionally, when ΦCL is small, the expansion of the membrane with increasing CL content was slower at higher surface pressure. These abnormal results are indicative of a folding structure being present before a collapsing structure, which was confirmed by using fluorescence microscopy and was characterized by using x-ray reflectivity with the electron density profile along the membrane's surface normal.

Origin of the Instability of Octadecylamine Langmuir Monolayer at Low pH.

It has been reported that an octadecylamine (ODA) Langmuir monolayer becomes unstable at low pH values with no measurable surface pressure at around pH 3.5, suggesting significant dissolution of the ODA molecule into the subphase solution (Albrecht, Colloids Surf. A 2006, 284-285, 166-174). However, by lowering the pH further, ODA molecules reoccupy the surface, and a full monolayer is recovered at pH 2.5. Using surface sum-frequency spectroscopy and pressure-area isotherms, it is found that the recovered monolayer at very low pH has a larger area per molecule with many gauche defects in the ODA molecules as compared to that at high pH values. This structural change suggests that the reappearance of the monolayer is due to the adsorbed Cl(-) counterions to the protonated amine groups, leading to partial charge neutralization. This proposition is confirmed by intentionally adding monovalent salts (i.e., NaCl, NaBr, or NaI) to the subphase to recover the monolayer at pH 3.5, in which the detailed structure of the monolayer is confirmed by sum frequency spectra and the adsorbed anions by X-ray reflectivity.

The fabrication of patterned gold nanoparticle arrays via selective ion irradiation and plasma treatment.

A simple and facile method for the patterning of gold nanoparticles (GNPs) was described via selective ion irradiation and oxygen plasma etching. Thin Pluronic films containing HAuCI4 as the precursor of GNPs were selectively irradiated through a pattern mask with 200 keV proton ions to generate GNP-embedded Pluronic patterns. The Pluronic was then removed by an oxygen plasma etching process for the pattern formation of GNPs. Based on the results of the UV-Vis, FE-SEM, and EDX analyses, 50 µm negative-tone line patterns of the GNP-embedded Pluronic were successfully generated at a fluence of less than 1 x 10(16) ions/cm2. The changes in the morphology and elemental composition of the formed GNP-embedded Pluronic patterns with different time periods of oxygen plasma etching were investigated using an FE-SEM with an EDX. The experimental results demonstrated that the patterns of GNPs were effectively generated by the oxygen plasma etching of the formed GNP-embedded Pluronic patterns for 15 min. Furthermore, the XRD results revealed that GNPs in the patterns formed by ion irradiation were further grown during the subsequent oxygen plasma etching.

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.

Topical Nanoliposomal Collagen Delivery for Targeted Fibril Formation by Electrical Stimulation.

Collagen is a complex, large protein molecule that presents a challenge in delivering it to the skin due to its size and intricate structure. However, conventional collagen delivery methods are either invasive or may affect the protein's structural integrity. This study introduces a novel approach involving the encapsulation of collagen monomers within zwitterionic nanoliposomes, termed Lip-Cols, and the controlled formation of collagen fibrils through electric fields (EF) stimulation. The results reveal the self-assembly process of Lip-Cols through electroporation and a pH gradient change uniquely triggered by EF, leading to the alignment and aggregation of Lip-Cols on the electrode interface. Notably, Lip-Cols exhibit the capability to direct the orientation of collagen fibrils within human dermal fibroblasts. In conjunction with EF, Lip-Cols can deliver collagen into the dermal layer and increase the collagen amount in the skin. The findings provide novel insights into the directed formation of collagen fibrils via electrical stimulation and the potential of Lip-Cols as a non-invasive drug delivery system for anti-aging applications.