Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Monitoring correlates of SARS-CoV-2 infection in cell culture using a two-photon-active calcium-sensitive dye.

BACKGROUND: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.

Tunable 2D Conjugated Porous Organic Polymer Films for Precise Molecular Nanofiltration and Optoelectronics.

Structural design of 2D conjugated porous organic polymer films (2D CPOPs), by tuning linkage chemistries and pore sizes, provides great adaptability for various applications, including membrane separation. Here, four free-standing 2D CPOP films of imine- or hydrazone-linked polymers (ILP/HLP) in combination with benzene (B-ILP/HLP) and triphenylbenzene (TPB-ILP/HLP) aromatic cores are synthesized. The anisotropic disordered films, composed of polymeric layered structures, can be exfoliated into ultrathin 2D-nanosheets with layer-dependent electrical properties. The bulk CPOP films exhibit structure-dependent optical properties, triboelectric nanogenerator output, and robust mechanical properties, rivaling previously reported 2D polymers and porous materials. The exfoliation energies of the 2D CPOPs and their mechanical behavior at the molecular level are investigated using density function theory (DFT) and molecular dynamics (MD) simulations, respectively. Exploiting the structural tunability, the comparative organic solvent nanofiltration (OSN) performance of six membranes having different pore sizes and linkages to yield valuable trends in molecular weight selectivity is investigated. Interestingly, the OSN performances follow the predicted transport modeling values based on theoretical pore size calculations, signifying the existence of permanent porosity in these materials. The membranes exhibit excellent stability in organic solvents at high pressures devoid of any structural deformations, revealing their potential in practical OSN applications.

A Molecular Hybrid of the GFP Chromophore and 2,2'-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy.

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.

A GFP Inspired 8-Methoxyquinoline-Derived Fluorescent Molecular Sensor for the Detection of Zn2+ by Two-Photon Microscopy.

An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two-photon cross-section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.

Conductive nanofiltration membranes via in situ PEDOT-polymerization for electro-assisted membrane fouling mitigation.

Nanofiltration (NF) membranes play a pivotal role in water treatment; however, the persistent challenge of membrane fouling hampers their stable application. This study introduces a novel approach to address this issue through the creation of a poly(3,4-ethylenedioxythiophene) (PEDOT)-based conductive membrane, achieved by synergistically coupling interfacial polymerization (IP) with in situ self-polymerization of EDOT. During the IP reaction, the concurrent generation of HCl triggers the protonation of EDOT, activating its self-polymerization into PEDOT. This interwoven structure integrates with the polyamide network to establish a stable selective layer, yielding a remarkable 90 % increase in permeability to 20.4 L m-2 h-1 bar-1. Leveraging the conductivity conferred by PEDOT doping, an electro-assisted cleaning strategy is devised, rapidly restoring the flux to 98.3 % within 5 min, outperforming the 30-minute pure water cleaning approach. Through simulations in an 8040 spiral-wound module and the utilization of the permeated salt solution for cleaning, the electro-assisted cleaning strategy emerges as an eco-friendly solution, significantly reducing water consumption and incurring only a marginal electricity cost of 0.055 $ per day. This work presents an innovative avenue for constructing conductive membranes and introduces an efficient and cost-effective electro-assisted cleaning strategy to effectively combat membrane fouling.

Asymmetric Membrane Capacitive Deionization Using Anion-Exchange Membranes Based on Quaternized Polymer Blends.

Membrane capacitive deionization (MCDI) for water desalination is an innovative technique that could help to solve the global water scarcity problem. However, the development of the MCDI field is hindered by the limited choice of ion-exchange membranes. Desalination by MCDI removes the salt (solute) from the water (solvent); this can drastically reduce energy consumption compared to traditional desalination practices such as distillation. Herein, we outline the fabrication and characterization of quaternized anion-exchange membranes (AEMs) based on polymer blends of polyethylenimine (PEI) and polybenzimidazole (PBI) that provides an efficient membrane for MCDI. Flat sheet polymer membranes were prepared by solution casting, heat treatment, and phase inversion, followed by modification to impart anion-exchange character. Scanning electron microscopy (SEM), atomic force microscopy (AFM), nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR) spectroscopy were used to characterize the morphology and chemical composition of the membranes. The as-prepared membranes displayed high ion-exchange capacity (IEC), hydrophilicity, permselectivity and low area resistance. Due to the addition of PEI, the high density of quaternary ammonium groups increased the IEC and permselectivity of the membranes, while reducing the area resistance relative to pristine PBI AEMs. Our PEI/PBI membranes were successfully employed in asymmetric MCDI for brackish water desalination and exhibited an increase in both salt adsorption capacity (>3×) and charge efficiency (>2×) relative to membrane-free CDI. The use of quaternized polymer blend membranes could help to achieve greater realization of industrial scale MCDI.

Synthesis and spectroscopic characterization of novel GFP chromophore analogues based on aminoimidazolone derivatives.

In order to improve the fluorescence properties of the green fluorescent protein chromophore, p­HOBDI ((5­(4­hydroxybenzylidene)­2,3­dimethyl­3,5­dihydro­4H­imidazol­4­one), sixteen dihydroimidazolone derivates were synthesized from thiohydantoin and arylaldehydes. The synthesis developed is an efficient, novel, one-pot procedure. The study provides a detailed description of the spectroscopic characteristics of the newly synthesized compounds, using p­HOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than p­HOBDI, up to 28 times higher quantum yields. An experimental and theoretical investigation of the relationship of the fluorescence properties with the molecular structure was also carried out. A good correlation was found between the emission wavenumber and the Hammett constant of the functional group, which suggests the intermolecular charge transfer (ICT) mechanism between the aromatic groups.

Robust Covalently Cross-linked Polybenzimidazole/Graphene Oxide Membranes for High-Flux Organic Solvent Nanofiltration.

Robust, readily scalable, high-flux graphene oxide (GO) mixed matrix composite membranes were developed for organic solvent nanofiltration. Hydroxylated polybenzimidazole was synthesized by N-benzylation of polybenzimidazole with 4-(chloromethyl)benzyl alcohol, which was confirmed by FTIR and NMR spectroscopy. Flat-sheet composite membranes comprising of polybenzimidazoles and 1 or 2 wt % GO were fabricated via conventional blade coating and phase inversion. Subsequently, GO was covalently anchored to the hydroxyl groups of the polymer using a diisocyanate cross-linking agent. The even distribution of GO in the membranes was mapped by visible-light microscopy. Hydroxylation and incorporation of GO in the polymer matrix increased the permeance up to 45.2 ± 1.6 L m-2 h-1 bar-1 in acetone, nearly 5 times higher than the unmodified benchmark membrane. The enhancement in permeance from the addition of GO did not compromise the solute rejection. The composite membranes were found to be tight in seven organic solvents, having molecular weight cut-offs (MWCO) as low as 140 g mol-1. Permeance increased with increasing solvent polarity, while rejection of a 420 g mol-1 pharmaceutical remained over 93%. The covalent anchoring resulted in robust composite membranes that maintained constant performance over 14 days in a continuous cross-flow configuration.