Graphene Nanoribbon Hybridization of Zeolitic Imidazolate Framework Membranes for Intrinsic Molecular Separation.

Zeolitic imidazolate frameworks (ZIFs) are promising for gas separation membrane, but their molecular cut-off differs from that expected from its intrinsic aperture structure because of their flexibility. Herein, we introduced graphene nanoribbons (GNRs) to rigidify the ZIF framework. Because the sp2 edge of the GNRs induces strong anchoring effects, the modified layer can be rigidified. Particularly, when the GNRs were embedded and distributed in the ZIF-8 layer, an intrinsic aperture size of 3.4 Šwas observed, resulting in high H2 /CO2 separation (H2 permeance: 5.2×10-6  mol/m2 Pa s, ideal selectivity: 142). The performance surpasses the upper bound of polycrystalline MOF membrane performance. In addition, the membrane can be applied to blue H2 production, as demonstrated with a simulated steam reformed gas containing H2 /CO2 /CH4 . The separation performance was retained in the presence of water. The fundamentals of the molecular transport through the rigid ZIF-8 framework were revealed using molecular dynamics simulations.

Lipid-Oriented Live-Cell Distinction of B and T Lymphocytes.

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.

Holding-Oriented versus Gating-Oriented Live-Cell Distinction: Highlighting the Role of Transporters in Cell Imaging Probe Development.

Small molecule imaging probes are powerful tools to understand complex biological systems. The mainstreams of imaging probe developments have been focused on the target holding of the probes; the holding targets are often cell-type-specific biomarkers. This type of the probe mechanism can be designated as holding-oriented live-cell distinction (HOLD). Our group has worked on the development of cell-type-selective probes using a diversity-oriented fluorescence library approach (DOFLA), where unbiased phenotypic screening is employed using fluorescent library compounds. Through the conventional target identification methods such as an affinity-based analysis, we elucidated that some of the probe mechanisms are HOLD. However, we also realized that sometimes there is no specific holding target for probes or the holding targets are ubiquitous. The observation led us to test an alternative mechanism of cell-type-specific probes as gating-oriented live-cell distinction (GOLD). We started to examine the gating mechanism of probes, which is mainly based on transporters but which does not necessarily require probe holding to cellular targets. Transporters can control the in and out movement of various nutrients and chemicals. Different expression levels of transporters in various cell types could provide the molecular mechanism of differential staining of cells by regulating the intracellular accumulation of a certain specific probe. A number of GOLD probes have been developed by modifying or mimicking endogenous substrates of transporters such as inorganic ions, glucose, amino acids, or neurotransmitters, utilizing broad substrate specificity of transporters. The radiolabeled or fluorophore-conjugated substrate mimetics have been widely used for live cell distinction and various applications such as disease-related cell or tissue imaging. In humans, there are about 400 solute carrier (SLC) transporters and 50 ATP-binding cassette (ABC) transporters. Since some transporters have broad substrate specificity, they can transport not only derivatives of endogenous natural substrates but also totally synthetic diverse imaging probes, such as DOFLA probes. Without preconsidering the structure of endogenous substrates, we recently demonstrated a series of live-cell imaging probes and elucidated their molecular mechanism as a gating one, either by SLC or ABC transporters. Transporter inhibitor panel and CRISPR-based transporter libraries could provide a systematic gating target elucidation platform. Considering the generality of DOFLA and the CRISPR-based genomic tool for transporter systems (>450 in humans), the GOLD approach will offer new insight and promise for unprecedented levels of novel cell imaging probe development.

Neural Tissue-Like, not Supraphysiological, Electrical Conductivity Stimulates Neuronal Lineage Specification through Calcium Signaling and Epigenetic Modification.

Electrical conductivity is a pivotal biophysical factor for neural interfaces, though optimal values remain controversial due to challenges isolating this cue. To address this issue, conductive substrates made of carbon nanotubes and graphene oxide nanoribbons, exhibiting a spectrum of conductivities from 0.02 to 3.2 S m-1, while controlling other surface properties is designed. The focus is to ascertain whether varying conductivity in isolation has any discernable impact on neural lineage specification. Remarkably, neural-tissue-like low conductivity (0.02-0.1 S m-1) prompted neural stem/progenitor cells to exhibit a greater propensity toward neuronal lineage specification (neurons and oligodendrocytes, not astrocytes) compared to high supraphysiological conductivity (3.2 S m-1). High conductivity instigated the apoptotic process, characterized by increased apoptotic fraction and decreased neurogenic morphological features, primarily due to calcium overload. Conversely, cells exposed to physiological conductivity displayed epigenetic changes, specifically increased chromatin openness with H3acetylation (H3ac) and neurogenic-transcription-factor activation, along with a more balanced intracellular calcium response. The pharmacological inhibition of H3ac further supported the idea that such epigenetic changes might play a key role in driving neuronal specification in response to neural-tissue-like, not supraphysiological, conductive cues. These findings underscore the necessity of optimal conductivity when designing neural interfaces and scaffolds to stimulate neuronal differentiation and facilitate the repair process.

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group.

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, Met(N-terminal), and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, Met(N-terminal)-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, Met(N-terminal), and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.

Graphene Nanoribbon/Carbon Nanotube Hybrid Hydrogel: Rheology and Membrane for Ultrafast Molecular Diafiltration.

Hybrids based on carbon nanotubes (CNTs) and graphene nanoribbons (GNRs) are expected to have synergistic effects for various applications. Herein, we demonstrate a simple one-pot synthesis of a CNT/GNR hybrid material by adjusting the oxidation and unzipping conditions of multi-walled CNTs (MWNTs). The MWNT/graphene oxide nanoribbon (GONR) hybrid was dispersed in various solvents, particularly showing the hybrid hydrogel phase in water at a concentration of 40 mg mL-1. The MWNT/GONR hydrogel exhibited shear-thinning behavior, which can be beneficial for coating a large-area MWNT/GONR layer onto a polymeric porous support by using a scalable slot-die coater. The MWNT/GONR membrane exhibited an outstanding nanofiltration performance, with a molecular weight cutoff of 300 Da and a dye/salt diafiltration performance with a separation factor of 1000 and a water flux of 367.8 LMH, far surpassing the upper bound of diafiltration performance of the existing membranes.

Fabrication Techniques for Graphene Oxide-Based Molecular Separation Membranes: Towards Industrial Application.

Graphene oxide (GO) has been a prized material for fabricating separation membranes due to its immense potential and unique chemistry. Despite the academic focus on GO, the adoption of GO membranes in industry remains elusive. One of the challenges at hand for commercializing GO membranes lies with large-scale production techniques. Fortunately, emerging studies have acknowledged this issue, where many have aimed to deliver insights into scalable approaches showing potential to be employed in the commercial domain. The current review highlights eight physical methods for GO membrane fabrication. Based on batch-unit or continuous fabrication, we have further classified the techniques into five small-scale (vacuum filtration, pressure-assisted filtration, spin coating, dip coating, drop-casting) and three large-scale (spray coating, bar/doctor blade coating, slot die coating) approaches. The continuous nature of the large-scale approach implies that the GO membranes prepared by this method are less restricted by the equipment's dimensions but rather the availability of the material, whereas membranes yielded by small-scale methods are predominately limited by the size of the fabrication device. The current review aims to serve as an initial reference to provide a technical overview of preparing GO membranes. We further aim to shift the focus of the audience towards scalable processes and their prospect, which will facilitate the commercialization of GO membranes.

Sonosensitizer-Functionalized Graphene Nanoribbons for Adhesion Blocking and Sonodynamic Ablation of Ovarian Cancer Spheroids.

Advanced stage ovarian cancer is challenging to treat due to widespread seeding of tumor spheroids throughout the mesothelial lining of the peritoneal cavity. In this work, a therapeutic strategy using graphene nanoribbons (GNR) functionalized with 4-arm polyethylene glycol (PEG) and chlorin e6 (Ce6), a sonosensitizer, to target metastatic ovarian cancer spheroids is reported. GNR-PEG-Ce6 adsorbs onto the spheroids and disrupts their adhesion to extracellular matrix proteins or LP-9 mesothelial cells. Furthermore, for spheroids that do adhere, GNR-PEG-Ce6 delays spheroid disaggregation and spreading as well as mesothelial clearance, key metastatic processes following adhesion. Owing to the sonodynamic effects of Ce6 and its localized delivery via the biomaterial, GNR-PEG-Ce6 can kill ovarian cancer spheroids adhered to LP-9 cell monolayers when combined with mild ultrasound irradiation. The interaction with GNR-PEG-Ce6 also loosens cell-cell adhesions within the spheroids, rendering them more susceptible to treatment with the chemotherapeutic agents cisplatin and paclitaxel, which typically have difficulty in penetrating ovarian cancer spheroids. Thus, this material can facilitate effective chemotherapeutic and sonodynamic combination therapies. Finally, the adhesion inhibiting and sonodynamic effects of GNR-PEG-Ce6 are also validated with tumor spheroids derived from the ascites fluid of ovarian cancer patients, providing evidence of the translational potential of this biomaterial approach.

Ultrafast H2-selective nanoporous multilayer graphene membrane prepared by confined thermal annealing.

H2 selective dense pores are generated in a graphene oxide (GO) layer by thermal-decomposition of oxygen-functional groups under high pressure. The nanoporous GO membrane shows H2/CO2 selectivity of 12.1 and H2 permeability of 10360 Barrer.

Diamine vapor treatment of viscoelastic graphene oxide liquid crystal for gas barrier coating.

A layered graphene oxide/ethylenediamine (GO/EDA) composite film was developed by exposing aqueous GO liquid crystal (GOLC) coating to EDA vapor and its effects on the gas barrier performance of GO film were systematically investigated. When a GO/EDA coating with a thickness of approximately 1 µm was applied to a neat polyethylene terephthalate (PET) film, the resultant film was highly impermeable to gas molecules, particularly reducing the gas permeance up to 99.6% for He and 98.5% for H2 in comparison to the neat PET film. The gas barrier properties can be attributed to the long diffusion length through stacked GO nanosheets. The EDA can crosslink oxygen-containing groups of GO, enhancing the mechanical properties of the GO/EDA coating with hardness and elastic modulus values up to 1.14 and 28.7 GPa, respectively. By the synergistic effect of the viscoelastic properties of GOLC and the volatility of EDA, this coating method can be applied to complex geometries and EDA intercalation can be spontaneously achieved through the scaffold of the GOLC.

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging.

Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.

Graphene Oxide Nanoribbon Hydrogel: Viscoelastic Behavior and Use as a Molecular Separation Membrane.

The preparation of carbon materials based hydrogels and their viscoelastic properties are essential for their broad application and scale-up. However, existing studies are mainly focused on graphene derivatives and carbon nanotubes, and the behavior of graphene nanoribbon (GNR), a narrow strip of graphene, remains elusive. Herein, we demonstrate the concentration-driven gelation of oxidized GNR (graphene oxide nanoribbon, GONR) in aqueous solvents. Exfoliated individual GONRs sequentially assemble into strings (∼1 mg/mL), nanoplates (∼20 mg/mL), and a macroporous scaffold (50 mg/mL) with increasing concentration. The GONR hydrogels exhibit viscoelastic shear-thinning behavior and can be shear-coated to form large-area GONR films on substrates. The entangled and stacked structure of the GONR film contributed to outstanding nanofiltration performance under high pressure, cross-flow, and long-term filtration, while the precise molecular separation with 100% rejection rate was maintained for sub-nanometer molecules.