Molecular assembly of carbon nitride-based composite membranes for photocatalytic sterilization and wound healing.

Polymeric carbon nitride (pCN) has attracted increasing interest as a metal-free photocatalyst because of its high efficiency in reactive oxygen species (ROS) generation. However, due to poor solubility, compounding pCN at the molecular level into more advanced nanocomposites remains a challenge. Herein, we report the dissolution of pCN in polyphosphoric acid (PPA) for the first time and fluid-phase assembly with carbon nanotubes (CNTs) into a flexible free-standing membrane. Mechanism and generality studies disclosed that the coordination of the acidity, viscosity, and adsorption energy of the solvents led to the successful dissolution of pCN. Interestingly, the pCN/CNTs molecular composite membrane exhibited not only superior mechanical properties and cycling performance as a result of strengthened π-π interfacial interaction, but also outstanding inactivation of E. coli and S. aureus in sterilization and wound healing for laboratory mice via photogenerated oxygen radicals. It would open a new era of pCN for biomedical applications in molecular composite membranes, beyond the traditional solar fuel applications in powders.

Elucidating Electrocatalytic Oxygen Reduction Kinetics via Intermediates by Time-Dependent Electrochemiluminescence.

Facile evaluation of oxygen reduction reaction (ORR) kinetics for electrocatalysts is critical for sustainable fuel-cell development and industrial H2 O2 production. Despite great success in ORR studies using mainstream strategies, such as the membrane electrode assembly, rotation electrodes, and advanced surface-sensitive spectroscopy, the time and spatial distribution of reactive oxygen species (ROS) intermediates in the diffusion layer remain unknown. Using time-dependent electrochemiluminescence (Td-ECL), we report an intermediate-oriented method for ORR kinetics analysis. Owing to multiple ultrasensitive stoichiometric reactions between ROS and the ECL emitter, except for electron transfer numbers and rate constants, the potential-dependent time and spatial distribution of ROS were successfully obtained for the first time. Such exclusively uncovered information would guide the development of electrocatalysts for fuel cells and H2 O2 production with maximized activity and durability.

Advances in electrochemiluminescence luminophores based on small organic molecules for biosensing.

Electrochemiluminescence (ECL) has several advantages, such as a near-zero background signal, high sensitivity, wide dynamic range, simplicity, and is widely used for sensing, imaging, and single cell analysis. ECL luminophores are the key factors in the performance of various applications. Among various luminophores, small organic luminophores exhibit many intriguing features including good biocompatibility, facile modification, well-defined molecular structure, and sustainable raw materials, making small organic luminophores attractive for the use in the ECL field. Although many great achievements have been made in the synthesis of new small organic luminophores, solving various challenges, and expanding new applications, there are almost no comprehensive reviews on small organic ECL luminophores. In this review, we briefly introduce the advantages and emission mechanisms of small organic ECL luminophores, summarize the main types, molecular characteristics, and ECL properties of most existing small organic ECL luminophores, and present the important applications and design principles in sensors, imaging, single cell analysis, sterilization, and other fields. Finally, the challenges and outlook of organic ECL luminophores to be popularized in biosensing applications are also discussed.

Voltammetric Study and Modeling of the Electrochemical Oxidation Process and the Adsorption Effects of Luminol and Luminol Derivatives on Glassy Carbon Electrodes.

Luminol is one of the most widely used electrochemiluminescence (ECL) reagents, yet the detailed mechanism and kinetics of the electrochemical oxidation of luminol remain unclear. We propose a model that describes the electrochemical oxidation of luminol as multiple electron transfer reactions followed by an irreversible chemical reaction, and we applied a finite element method simulation to analyze the electron transfer kinetics in alkaline solutions. Although negligible at higher pH values, the adsorption of luminol on the glassy carbon electrode became noticeable in a solution with pH = 12. Additionally, various types of adsorption behaviors were observed for luminol derivatives and analogues, indicating that the molecular structure affected not only the oxidation but also the adsorption process. The adsorption effect was analyzed through a model with a Langmuir isotherm to show that the saturated surface concentration as well as the reaction kinetics increased with decreasing pH, suggesting a competition for the active sites between the molecule and OH-. Moreover, we show that the ECL intensity could be boosted through the adsorption effect by collecting the ECL intensity generated through the electrochemical oxidation of luminol and a luminol analogue, L012, in a solution with pH = 13. In contrast with luminol, a significant adsorption effect was observed for L012 at pH = 13, and the ECL intensity was enhanced by the adsorbed species, especially at higher scan rates. The magnitude of the enhancement of the ECL intensity matched well with the simulation using our model.

Extended Conjugation Tuning Carbon Nitride for Non-sacrificial H2 O2 Photosynthesis and Hypoxic Tumor Therapy.

Artificial photocatalysis offers a clean approach for producing H2 O2 . However, the poor selectivity and activity of H2 O2 production hamper traditional industrial applications and emerging photodynamic therapy (PDT)/chemodynamic therapy (CDT). Herein, we report a C5 N2 photocatalyst with a conjugated C=N linkage for selective and efficient non-sacrificial H2 O2 production in both normoxic and hypoxic systems. The strengthened delocalization of π-electrons by linkers in C5 N2 downshifted the band position, thermodynamically eliminating side H2 evolution reaction and kinetically promoting water oxidation. As a result, C5 N2 had a competitive solar-to-chemical conversion efficiency of 0.55 % in overall H2 O2 production and exhibited by far the highest activity under hypoxic conditions (698 µM h-1 ). C5 N2 was further applied to hypoxic PDT/CDT with outstanding performance in apparent cancer cell death and synchronous bioimaging. The study sheds light on the photosynthesis of H2 O2 by carbon nitrides for health applications.

Photoelectron Storages in Functionalized Carbon Nitrides for Colorimetric Sensing of Oxygen.

Colorimetric sensors have been widely used for centuries across diverse fields, thanks to their easy operation and uncompromisingly high sensitivity with no need for electricity. However, it is still a great challenge for conventional chromogenic systems to perform multiple measurements meanwhile maintaining high robustness. Here, we reported that carbon nitrides (CNs), the raw materials that are abundant, structure-tunable, and stable semiconductors with photoelectron storage capability, can be developed as a chromogenic system for colorimetric sensors. Beyond conventional metal oxides that only demonstrated a single blue-color switch after photoelectron storage, CN exhibited a multicolor switch under identical conditions owing to the unusual multiple photoelectron storage pathways. Mechanism studies revealed cyano and carbonyl groups in CN crucially elongated the centroid distance of electrons/holes, which exclusively stabilized the specific excited states that have different light absorption; meanwhile, the counter cations strengthened these processes. As a result, O2, a proof-of-concept analyte, was quantitatively detected by the CN-derived colorimetric sensor, showing high reversibility in hundreds of cycles and adaptable sensitivity/detection range, outperforming most reported and commercial oxygen sensors. These intriguing features of CN are highly envisioned for the next generation of colorimetric sensors, especially in developing countries or fieldworks, to improve the detection reliability and lower the sensing cost.

Lighting Up Electrochemiluminescence-Inactive Dyes via Grafting Enabled by Intramolecular Resonance Energy Transfer.

Due to near-zero optical background and photobleaching, electrochemiluminescence (ECL), an optical phenomenon excited by electrochemical reactions, has drawn extensive attention, especially for ultrasensitive bioassays. Developing diverse ECL emitters is crucial to unlocking their multiformity and performances but remains a formidable challenge due to the rigorous requirements for ECL. Herein, we report a general strategy to light up ECL-inactive dyes in an aqueous solution via grafting, a well-developed concept for plant propagation since 500 BCE. As a proof of concept, a series of luminol donor-dye acceptor-based ECL emitters were grafted with near-unity resonance energy transfer (RET) efficiency and coarse/fine-tunable emission wavelengths. Rather than the sophisticated design of new skeleton-based molecules to meet all of the prerequisites for ECL in a constrained manner, each unit in the proposed ECL ensemble performed its functions maximally. As a result, beyond traditional two-dimensional (2D) ones, a three-dimensional (3D) coordinate biosensing system, simultaneously showing a calibration curve and selectivity, was established using the new ECL emitter. This lighting up strategy would generally address the scarcity of ECL emitters and enable unprecedented functions.

A methyltransferase-like 14/miR-99a-5p/tribble 2 positive feedback circuit promotes cancer stem cell persistence and radioresistance via histone deacetylase 2-mediated epigenetic modulation in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and treatment-resistant tumor. The biological implications and molecular mechanism of cancer stem-like cells (CSCs) in ESCC, which contribute to therapeutic resistance such as radioresistance, remain elusive. METHODS: Quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and in situ hybridization assays were used to detect methyltransferase-like 14 miR-99a-5p tribble 2 (METTL14/miR-99a-5p/TRIB2) expression in ESCC. The biological functions of METTL14/miR-99a-5p/TRIB2 were demonstrated in vitro and in vivo. Mass spectrum analysis was used to identify the downstream proteins regulated by TRIB2. Chromatin immunoprecipitation (IP), IP, N6 -methyladenosine (m6 A)-RNA IP, luciferase reporter, and ubiquitination assays were employed to explore the molecular mechanisms underlying this feedback circuit and its downstream pathways. RESULTS: We found that miR-99a-5p was significantly decreased in ESCC. miR-99a-5p inhibited CSCs persistence and the radioresistance of ESCC cells, and miR-99a-5p downregulation predicted an unfavorable prognosis of ESCC patients. Mechanically, we unveiled a METTL14-miR-99a-5p-TRIB2 positive feedback loop that enhances CSC properties and radioresistance of ESCC cells. METTL14, an m6 A RNA methyltransferase downregulated in ESCC, suppresses TRIB2 expression via miR-99a-5p-mediated degradation of TRIB2 mRNA by targeting its 3' untranslated region, whereas TRIB2 induces ubiquitin-mediated proteasomal degradation of METTL14 in a COP1-dependent manner. METTL14 upregulates miR-99a-5p by modulating m6 A-mediated, DiGeorge critical region 8-dependent pri-mir-99a processing. Hyperactivation of TRIB2 resulting from this positive circuit was closely correlated with radioresistance and CSC characteristics. Furthermore, TRIB2 activates HDAC2 and subsequently induces p21 epigenetic repression through Akt/mTOR/S6K1 signaling pathway activation. Pharmacologic inhibition of HDAC2 effectively attenuates the TRIB2-mediated effect both in vitro and in patient-derived xenograft models. CONCLUSION: Our data highlight the presence of the METTL14/miR-99a-5p/TRIB2 axis and show that it is positively associated with CSC characteristics and radioresistance of ESCC, suggesting potential therapeutic targets for ESCC treatment.

N6-methyladenosine-modified circIGF2BP3 inhibits CD8+ T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer.

BACKGROUND: An in-depth understanding of immune evasion mechanisms in tumors is crucial to overcome resistance and enable innovative advances in immunotherapy. Circular RNAs (circRNAs) have been implicated in cancer progression. However, much remains unknown regarding whether circRNAs impact immune escape in non-small-cell lung carcinoma (NSCLC). METHODS: We performed bioinformatics analysis to profile and identify the circRNAs mediating immune evasion in NSCLC. A luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pulldown assays and fluorescence in situ hybridization were performed to identify the interactions among circIGF2BP3, miR-328-3p, miR-3173-5p and plakophilin 3 (PKP3). In vitro T cell-mediated killing assays and in vivo syngeneic mouse models were used to investigate the functional roles of circIGF2BP3 and its downstream target PKP3 in antitumor immunity in NSCLC. The molecular mechanism of PKP3-induced PD-L1 upregulation was explored by immunoprecipitation, RIP, and ubiquitination assays. RESULTS: We demonstrated that circIGF2BP3 (hsa_circ_0079587) expression was increased in NSCLC and negatively correlated with CD8+ T cell infiltration. Functionally, elevated circIGF2BP3 inactivated cocultured T cells in vitro and compromised antitumor immunity in an immunocompetent mouse model, and this effect was dependent on CD8+ T cells. Mechanistically, METTL3 mediates the N6-methyladenosine (m6A) modification of circIGF2BP3 and promotes its circularization in a manner dependent on the m6A reader protein YTHDC1. circIGF2BP3 competitively upregulates PKP3 expression by sponging miR-328-3p and miR-3173-5p to compromise the cancer immune response. Furthermore, PKP3 engages with the RNA-binding protein FXR1 to stabilize OTUB1 mRNA, and OTUB1 elevates PD-L1 abundance by facilitating its deubiquitination. Tumor PD-L1 deletion completely blocked the impact of the circIGF2BP3/PKP3 axis on the CD8+ T cell response. The inhibition of circIGF2BP3/PKP3 enhanced the treatment efficacy of anti-PD-1 therapy in a Lewis lung carcinoma mouse model. Collectively, the PKP3/PD-L1 signature and the infiltrating CD8+ T cell status stratified NSCLC patients into different risk groups. CONCLUSION: Our results reveal the function of circIGF2BP3 in causing immune escape from CD8+ T cell-mediated killing through a decrease in PD-L1 ubiquitination and subsequent proteasomal degradation by stabilizing OTUB1 mRNA in a PKP3-dependent manner. This work sheds light on a novel mechanism of PD-L1 regulation in NSCLC and provides a rationale to enhance the efficacy of anti-PD-1 treatment in NSCLC.

Bound oxygen-atom transfer endows peroxidase-mimic M-N-C with high substrate selectivity.

Advances in nanoscience have stimulated the wide exploration of nanozymes as alternatives to enzymes. Nonetheless, nanozymes often catalyze multiple reactions and are not specialized to a specific substrate, restricting their broad application. Here, we report that the substrate selectivity of the peroxidase-mimic M-N-C can be significantly altered via forming bound intermediates with variable interactions with substrates according to the type of metal. Taking two essential reactions in chemical sensing as an example, Fe-N-C and Co-N-C showed opposite catalytic selectivity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and 3-aminophthalhydrazide (luminol), respectively, by factors of up to 200-fold. It was revealed that specific transition metal-N coordination was the origin of the selective activation of H2O2 forming critically bound oxygen intermediates (M[double bond, length as m-dash]O) for oxygen-atom transfer and the consequent oxidization of substrates. Notably, owing to the embedded ligands in the rigid graphitic framework, surprisingly, the selectivity of M-N-C was even superior to that of commonly used horseradish peroxidase (HRP).

IFI6 depletion inhibits esophageal squamous cell carcinoma progression through reactive oxygen species accumulation via mitochondrial dysfunction and endoplasmic reticulum stress.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal forms of adult cancer with poor prognosis. Substantial evidence indicates that reactive oxygen species (ROS) are important modulators of aggressive cancer behavior. However, the mechanism by which ESCC cells integrate redox signals to modulate carcinoma progression remains elusive. METHODS: The expression of interferon alpha inducible protein 6 (IFI6) in clinical ESCC tissues and cell lines was detected by RT-PCR and Western blotting. The correlation between IFI6 expression levels and aggressive ESCC disease stage was examined by immunohistochemistry. Bioinformatic analysis was conducted to explore the potential function of IFI6 in ESCC. ESCC cell lines stably depleted of IFI6 and ectopically expressing IFI6 were established using lentiviruses expressing shRNAs and an IFI6 expression plasmid, respectively. The effects of IFI6 on ESCC cells were determined by cell-based analyses, including EdU assay, apoptotic assay, cellular and mitochondria-specific ROS detection, seahorse extracellular flux, and mitochondrial calcium flux assays. Blue native-polyacrylamide gel electrophoresis was used to determine mitochondrial supercomplex assembly. Transcriptional activation of NADPH oxidase 4 (NOX4) via ATF3 was confirmed by dual luciferase assay. In vivo tumor growth was determined in mouse xenograft models. RESULTS: We find that the expression of IFI6, an IFN-stimulated gene localized in the inner mitochondrial membrane, is markedly elevated in ESCC patients and a panel of ESCC cell lines. High IFI6 expression correlates with aggressive disease phenotype and poor prognosis in ESCC patients. IFI6 depletion suppresses proliferation and induces apoptosis by increasing ROS accumulation. Mechanistically, IFI6 ablation induces mitochondrial calcium overload by activating mitochondrial Ca2+ uniporter and subsequently ROS production. Following IFI6 ablation, mitochondrial ROS accumulation is also induced by mitochondrial supercomplex assembly suppression and oxidative phosphorylation dysfunction, while IFI6 overexpression produces the opposite effects. Furthermore, energy starvation induced by IFI6 inhibition drives endoplasmic reticulum stress through disrupting endoplasmic reticulum calcium uptake, which upregulates NOX4-derived ROS production in an ATF3-dependent manner. Finally, the results in xenograft models of ESCC further corroborate the in vitro findings. CONCLUSION: Our study unveils a novel redox homeostasis signaling pathway that regulates ESCC pathobiology and identifies IFI6 as a potential druggable target in ESCC.

Ultrafast Condensation of Carbon Nitride on Electrodes with Exceptional Boosted Photocurrent and Electrochemiluminescence.

Semiconducting polymeric carbon nitride (CN) has drawn wide attention ranging from photocatalysis to more recent biosensing owing to unique defect-tolerated optoelectronic properties and being metal-free, cheap, and highly stable. However, at the core of electrical-optical interconversion, the preparation of the CN photoelectrode is still challenging. Now, the growth of CN on electrodes is achieved simply by microwave-assisted condensation in seconds. The ultrafast heating not only addressed the thermodynamic contradiction of precursor volatilization during polymerization but also led to strongly adhesive CN layer on electrodes with gradient carbon-rich texture, greatly accelerating the electron-hole separation and mobility. Consequently, the CN photoelectrode exhibited a remarkable photocurrent and a record cathodic efficiency of electrochemiluminescence up to 7 times that of benchmark Ru(bpy)3 Cl2 in aqueous solution.

Exfoliation and Sensitization of 2D Carbon Nitride for Photoelectrochemical Biosensing under Red Light.

Two-dimensional carbon nitride (CN) has drawn increasing attention as a conjugated metal-free polymer for photoelectrochemical (PEC) biosensing. However, CN only absorbs ultraviolet and very limited visible light (λ<460 nm), which poses potential risks for biomolecules and also cannot pass through tissue for in vivo detection. Herein, simultaneous exfoliation and functionalization of CN nanosheets (CNNS) with copper phthalocyanine (TsCuPc) simply by mechanical milling, thanks to the delicate π-π interaction between them, is reported. Moreover, due to energy-level matching, an effective donor-acceptor (D-A) interaction with much-improved photocurrent under irradiation with red light (λ>630 nm) was observed for the as-prepared CNNS-TsCuPc. As an example, dopamine in blood was detected by using red light by a CNNS-TsCuPc photoelectrode with uncompromised linear range and detection limit, as well high selectivity. As one of the few successful demonstrations of red-light-responsive PEC sensing systems, this work takes a first step toward future in vivo applications by enriching the optoelectronic properties of CN with task-specific antenna molecules via D-A interaction.

Hot-Tailoring of Carbon Nitride Dots with Redshifted Photoluminescence for Visual Double Text Encryption and Bioimaging.

The fabrication of carbon dots and their doped forms by top-down chemical cleavage has attracted considerable attention in the efforts to meet the increasing demands for optoelectronic applications ranging from biosensing to electro- and photocatalysis. However, due to strong quantum confinement effects, the size decrease often leads to an increase in the band gap, even in the emission of deep-ultraviolet (DUV) light, which greatly limits their applications. Here, we report a facile hot-tailoring strategy for fabricating carbon nitride nanodots (CNDs) with redshifted intrinsic photoluminescent (PL) emission, compared with the pristine bulk precursor. It has been found that the different leaving abilities of the C,N-containing groups during the pyrolysis stage and the chemical passivation during the liquid-collection stage played vital roles. Due to the redshifted photoluminescence and other attractive features, the as-obtained CNDs were successfully applied in visual double text encryption with higher security and also in bioimaging with photostability superior to traditional dyes. This work highlights the great potential of the hot-tailoring method in modulating carbon-based nanostructures and offsetting band-gap widening as the size decreases.

Paper-based biosensor relying on flower-like reduced graphene guided enzymatically deposition of polyaniline for Pb(2+) detection.

A multi-amplified paper-based electrochemical strategy using Pb(2+) dependent DNAzyme as the recognition unit for Pb(2+) detection was developed. In this work, flower-like reduced graphene (FrGO) was prepared utilizing flower-like ZnO as template, which was first one step grown on the gold nanoparticles modified paper working electrode (Au-PWE). After being treated with acid and then modified with Au, a novel sensor platform named Au/FrGO/Au-PWE with large specific surface area and good electrical conductivity was fabricated. The Mn2O3 nanoparticle-assembled hierarchical hollow spheres (H-Mn2O3) was served as nanocarrier to immobilize GOx, HRP and signal strand (S3), resulting to the formation of S3/H-Mn2O3/HRP/GOx bioconjugations. In the presence of Pb(2+), the DNAzyme (S1) was activated and the substrate strand (S2) was cleaved. After the incubation with S3/H-Mn2O3/HRP/GOx in 0.1M HAc-NaAc solution (pH 4.3) containing 30 mM aniline and 15 mM glucose, a readily measurable "turn-on" electrochemical signal could be measured. On the basis of the signal amplification strategy of Au/FrGO/Au-PWE sensing platform and S3/H-Mn2O3/HRP/GOx bioconjugations, the developed biosensor exhibited a good linear response toward over a wide range of concentration from 0.005 to 2000 nM.

Paper-Based Analytical Devices Relying on Visible-Light-Enhanced Glucose/Air Biofuel Cells.

A strategy that combines visible-light-enhanced biofuel cells (BFCs) and electrochemical immunosensor into paper-based analytical devices was proposed for sensitive detection of the carbohydrate antigen 15-3 (CA15-3). The gold nanoparticle modified paper electrode with large surface area and good conductibility was applied as an effective matrix for primary antibodies. The glucose dehydrogenase (GDH) modified gold-silver bimetallic nanoparticles were used as bioanodic biocatalyst and signal magnification label. Poly(terthiophene) (pTTh), a photoresponsive conducting polymer, served as catalyst in cathode for the reduction of oxygen upon illumination by visible light. In the bioanode, electrons were generated through the oxidation of glucose catalyzed by GDH. The amount of electrons is determined by the amount of GDH, which finally depended on the amount of CA15-3. In the cathode, electrons from the bioanode could combine with the generated holes in the HOMO energy level of cathode catalysts pTTh. Meanwhile, the high energy level photoexcited electrons were generated in the LUMO energy level and involved in the oxygen reduction reaction, finally resulting in an increasing current and a decreasing overpotential. According to the current signal, simple and efficient detection of CA15-3 was achieved.