Enzyme-like Activity Research Articles

Dual-modal detection of antimicrobial susceptibility in pathogenic bacteria based on the high-throughput microfluidic platform

For pathogenic bacterial infectious diseases such as sepsis, accurate and rapid antimicrobial susceptibility testing (AST) plays an important role in improving antimicrobial screening and clinical outcomes. Traditional culture-based AST assays have the limitations of time-consuming, labor-intensive, expensive, and consuming reagents. In this work, a simple, fast, high-throughput microfluidic platform integrated with isolation, identification, and detection functions was designed for antibiotic susceptibility analysis in pathogenic bacteria. For the above purpose, MnO2@ZIF-90 nanoprobes with dual functions of nanozyme and fluorescence were prepared for visual eye or RGB analysis through a smartphone APP. More importantly, the redox activity of bacterial membrane in active pathogenic bacteria could decompose the MnO2 of MnO2@ZIF-90 nanoprobes and reduce the enzyme-like activity, which weakens the colorimetric signal of Timebox. Meanwhile, the ATP released from active pathogenic bacteria further triggered the breakdown of the inner ZIF-90 layer, generating a fluorescence signal. Therefore, a dual detection combining colorimetric and fluorescence was used to perform more accurate and reliable antibiotic susceptibility analysis of pathogenic bacteria. With the proposed microfluidic platform, a low detection of 10 CFU mL−1 bacteria and fast AST results were achieved within 5 min. The clinical applicability of the platform was further demonstrated through the analysis of clinical samples. This work provides a low-cost, easy-to-operation, fast-response, and high-throughput platform for the AST assays in pathogenic bacterial infectious diseases such as sepsis.

Bioorthogonal Regulated Metabolic Balance for Promotion of Ferroptosis and Mild Photothermal Therapy.

The nanozyme with NADPH oxidase (NOX)-like activity can promote the consumption of NADPH and the generation of free radicals. In consideration of that the upregulation of glucose-6-phosphate dehydrogenase (G6PD) would accelerate the compensation production of NADPH, for inhibition of G6PD activity, our designed bioorthogonal nanozyme can in situ catalyze pro-DHEA to produce G6PD inhibitor and dehydroepiandrosterone (DHEA) drugs to inhibit G6PD activity. Therefore, the well-defined platform can disrupt NADPH homeostasis, leading to the collapse of the antioxidant defense system in the tumor cells. The enzyme-like activity of PdCuFe is further enhanced when irradiated by NIR-II light. The destruction of NADPH homeostasis can promote ferroptosis and, in turn, facilitate mild photothermal therapy. Our design can realize NADPH depletion and greatly improve the therapeutic effect through metabolic regulation, which may provide inspiration for the design of bioorthogonal catalysis.

Organophosphorus Hydrolase-like Nanozyme with an Activity-Quenched Aggregation-Induced Emission Effect: A Self-Reporting and Specific Assay of Nerve Agents.

Given the promising prospect of aggregation-induced emission luminogens (AIEgens) in fluorescence assays, it is interesting and significant to endow AIEgens with molecular recognition capability (such as enzyme-like activity). Here, an AIE nanomaterial with intrinsic enzyme-like activity (named as "AIEzyme") is designed and synthesized via a facile coordination polymerization of Zr4+ and AIE ligands. AIEzyme possesses enhanced and stable fluorescence in different solvents because of the AIE effect of ligands in the rigid structure of a coordination polymer. On the other hand, the organophosphorus hydrolase (OPH)-mimicking activity of AIEzyme exhibits excellent affinity and specific activity. Interestingly, the OPH-like activity can quench the inherent fluorescence of AIEzyme by the hydrolysate of a typical organophosphorus nerve agent (OPNA), diethyl-4-nitrophenylphosphate. Due to the sensitive activity-induced quenching effect for AIE, the self-reporting fluorescence assay method based on AIEzyme was established, which shows ultrahigh sensitivity, high selectivity, good storage stability, and acceptable reliability for a real sample assay. Moreover, the simultaneous colorimetric method broadens the detection range and the application scenarios. The proposed assay method avoided the interference of O2 during detection because the OPH-like activity does not derive from the generation of ROS. As a bonus, AIEzyme can also be used for the degradation of OPNAs by OPH-like activity, and the process can be self-monitored by AIE quenching. This work would provide a new opportunity for expanding the application of AIEgens and artificial enzymes by endowing AIEgens with enzyme-like activity.

Spatiotemporally Guided Single-Atom Bionanozyme for Targeted Antibiofilm Treatment.

The heterogeneous and dynamic microenvironment of biofilms complicates bacterial infection treatment. Nanozyme catalytic therapy has recently been promising in treating biofilm infections. However, active nanozymes designed with the required precision targeting the biofilm microenvironment are lacking. This work proposes a spatiotemporally guided single-atom bionanozyme (BioSAzyme) for targeted antibiofilm therapy based on protein engineering of copper single-atom nanozyme (Cu SAzyme). The Cu SAzyme, synthesized via a novel mechanochemistry-assisted method, features highly accessible Cu-N4 active sites exposed on 2D N-doped carbon, exhibiting excellent triple enzyme-like activities according to experimental results and density functional theory calculations. Inheriting biofunctionality from both glucose oxidase and concanavalin A, BioSAzyme can localize the biofilm glycocalyx and catalyze endogenous glucose into H₂O₂ and gluconic acid, thus triggering multiplex cascade reactions with pH self-adaption to consume glucose and glutathione and generate •OH radicals. This spatiotemporally guided bionanocatalytic agent effectively inhibits E. coli O157: H7 and methicillin-resistant S. aureus biofilms in vitro and in vivo. Taking together, this work opens up new avenues for the rational design of single-atom nanozymes for precise antibiofilm therapy.

Engineering Injectable Coassembled Hydrogel by Photothermal Driven Chitosan-Stabilized MoS2 Nanosheets for Infected Wound Healing.

The application of enzyme-like molybdenum disulfide (MoS2) in tissue repair was confronted with stable dispersion, solubilization, and biotoxicity. Here, the injectable self-healing hydrogel was successfully designed using a step-by-step coassembly of chitosan and MoS2. Polyphenolic chitosan as a "structural stabilizer" of MoS2 nanosheets reconstructed well-dispersed MoS2@CSH nanosheets, which improved the biocompatibility of traditional MoS2, and strengthened its photothermal conversion and enzyme-like activities, guaranteeing highly efficient radical scavenging and antimicrobial properties. Furthermore, the polyphenol chitosan was employed again as a "molecular cross-linking agent" to form the injectable NIR-responsive MoS2@CSH hydrogel by accelerating hydrogen-bond interaction among chitosan and the multicross-linking reaction among polyphenols. The rapid self-healing ability was conducive to wound closure and dynamic adaptability. An experimental study on infected wound healing demonstrated that MoS2@CSH hydrogel could substantially eradicate bacteria and accelerate the angiogenesis of infected wounds. The photothermal-driven coassembly of MoS2 and polycation provided an alternative strategy for infected wound healing.

Chiral FeNC single-atom nanozymes with multi-enzyme activity for dye degradation

The application of single-atom nanozymes in the removal of organic dye pollution by advanced oxidation process (AOPs) have been widely concerned. However, the application of single-atom nanozymes in AOPs is rarely reported. In this work, a kind of Fe-and nitrogen-codoped carbon (Fe-N-C) with peroxidase-like and laccase-like activities was prepared by calcining Fe-doped MOF, then the chiral peptides were introduced into single-atom nanozymes, and chiral single-atom nanozymes (L-Au@FeNC) were successfully prepared. The L-Au@FeNC can effectively activate PMS to degrade different kinds of organic dyes and also shows high cycle stability. In addition, the results of quenching experiment and electron paramagnetic resonance (EPR) test show that 1O2 produced during PMS activation plays an important role in the degradation of organic dyes, and O2·-, ·OH, and SO4·- are also produced during PMS activation, suggesting that the degradation of selected organic dyes include radical and non-radical pathways. It should be noted that L-Au@FeNC showed higher activity than D-Au@FeNC in enzyme-like activity (laccase-like and peroxidase-like) experiment and organic dye degradation, the results of molecular docking simulation indicated that it may be due to the better binding force of L peptide to the substrate of enzyme-like reaction and organic dye than D peptide. This chiral single-atom nanozymes provide a new idea for the application of organic dye degradation and enzyme-like catalysis design.

Single-atom nanozymes possessing robust multienzyme-mimetic catalytic properties for sensing of volatile alkaline gas

Volatile alkaline gases (VAGs) emitted from various sources, such as the food or chemical industry, pose potential harm to the natural environment and human health. Therefore, the development of a real-time, rapid, and cost-effective detection method is crucial. In this study, the enzyme-like catalytic types and activities of four different metal-based single-atom enzymes (Ga, Cu, Mn, and Zn SAzymes) were selected and evaluated. Among them, Ga SAzyme exhibited the most promising catalytic properties. Furthermore, the multienzyme catalytic properties of Ga SAzyme were utilized for the detection of ammonia, and it was found that its peroxidase-like (POD-like) activity showed a strong affinity for ammonia (Km = 0.05 mM). This detection strategy demonstrated high sensitivity, with a limit of detection (LOD) of 3.0 mM in the linear range of 0.01–0.05 M and 7.0 mM in the linear range of 0.075–0.25 M. Additionally, the method was characterized by fast response time (15 s) and low cost ($0.035 per sample). The proposed method holds great potential for the detection of VAGs from various sources in the future.

Edible ultrasmall polyphenolic nanozymes for oral treatment of alcohol-induced acute gastritis

Excessive alcohol consumption and prolonged administration of non-steroidal anti-inflammatory drugs (NSAIDs) lead to gastritis which affects millions globally, yet safe and effective treatment options remain limited. While a range of nanozymes have been employed in the oral treatment of inflammatory bowel disease (IBD), the research directed toward gastritis therapy remains unexplored. The challenges of rapid gastric emptying, harsh gastric acid erosion, and the non-edible ingredient constitution hinder the practical use of nanozymes in this context. Herein, three types of sub-10 nm ultrasmall iron-polyphenol nanozymes (UIPNs) were synthesized by using fully edible natural active polyphenols, iron ions and PVP. UIPNs rapidly penetrated the gastric mucosa, resided in the stomach for over 12 h, and remained stable in the harsh acidic environment. They exhibited excellent enzyme-like activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). Compared to omeprazole, all three types of UIPNs showed superior activity, with Fis UIPNs demonstrating the best performance in preventing and protecting against alcohol-induced gastric mucosal injury in mice. The fully food-sourced components, constructed into ultrasmall nanozymes via non-covalent interactions, ensure long-term safety and hold promise for playing significant roles in various gastrointestinal diseases.

Flower-like boron-doped zinc single-atom nanozymes for colorimetric sensing and smartphone-assisted detection of Cr(VI)

With the development of electronics, electroplating, printing, and dyeing industries, environmental pollution caused by hexavalent chromium (Cr(VI)) has become increasingly prominent. Skin contact with Cr (VI) can cause allergies or genetic defects, and inhalation can cause cancer, which is a lasting danger to the environment and the human body. Developing effective strategies to monitor Cr(VI) in environmental water or industrial wastewater can evaluate the degree of water pollution and risk warning, thus helping to prevent the spread of Cr(VI) pollution, promote the protection of water resources and the ecological environment, and ensure human safety and sustainable development. On the basis of the regulation of dopamine, boron-doped zinc single-atom nanozymes (Zn/B-NC SAzymes) with three-dimensional nanoflower morphology were controlled in this work. The introduction of B in Zn/B-NC SAzymes and the high metal loading of Zn (6.5 wt%) led to the formation of more active sites, resulting in the material showing excellent enzyme-like activity. H2O2 decomposed to generate superoxide radicals under the catalysis of Zn/B-NC SAzymes, which then oxidized the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to generate blue oxTMB. When Cr(VI) was introduced into the sensor system, the color of blue oxTMB is deepened, and the colorimetric method of Cr(VI) was constructed. The linear range is 0.2–40 μM, LOD is 59 nM, and the visual detection of Cr (VI) is performed with the aid of the smartphone. This work not only provides experimental and theoretical guidance for understanding the active centers of Zn-SAzymes and their catalytic processes, but also provides a promising and alternative detection strategy for the rapid and even visual on-site detection of Cr(VI) in aquatic environments, which is of great significance for the control of Cr(VI) pollution in the environment and industrial wastewater.

Photothermal amplified multizyme activity for synergistic photothermal-catalytic tumor therapy

Nano-enzymatic catalytic therapy has been widely explored as a promising tumor therapeutic method with specific responsiveness to the tumor microenvironment (TME). However, the inherent lower and simplex catalytic efficiency impairs their anti-tumor efficacy. Therefore, developing novel nanozymes with relatively high and multiple catalytic characteristics, simultaneously enhancing the enzyme-like activity of nanozymes using the proper method, photothermal promoted catalytic property, is a reliable way. In this paper, we report a manganese oxide/nitrogen-doped carbon composite nanoparticles (MnO-N/C NPs) with multi-enzyme mimetic activity and photothermal conversional effect. The peroxidase (POD)-like/oxidase (OXD)-like/catalase (CAT)-like activity of MnO-N/C nanozymes was accelerated upon exposure to an 808 nm NIR laser. In vitro and in vivo results proved that the MnO-N/C NPs shown excellent magnetic resonance imaging (MRI) guided synergistic photothermal-enhanced catalytic treatment and photothermal therapy of liver cancer. The photothermal enhanced multi-enzyme activity maximizes the efficacy of catalytic and photothermal therapy while reducing harm to healthy cells, thereby offering valuable insights for the development of next-generation photothermal nanozymes to enhance tumor therapy.

Atomic-scale strain engineering of atomically resolved Pt clusters transcending natural enzymes

Strain engineering plays an important role in tuning electronic structure and improving catalytic capability of biocatalyst, but it is still challenging to modify the atomic-scale strain for specific enzyme-like reactions. Here, we systematically design Pt single atom (Pt1), several Pt atoms (Ptn) and atomically-resolved Pt clusters (Ptc) on PdAu biocatalysts to investigate the correlation between atomic strain and enzyme-like catalytic activity by experimental technology and in-depth Density Functional Theory calculations. It is found that Ptc on PdAu (Ptc-PA) with reasonable atomic strain upshifts the d-band center and exposes high potential surface, indicating the sufficient active sites to achieve superior biocatalytic performances. Besides, the Pd shell and Au core serve as storage layers providing abundant energetic charge carriers. The Ptc-PA exhibits a prominent peroxidase (POD)-like activity with the catalytic efficiency (Kcat/Km) of 1.50 × 109 mM−1 min−1, about four orders of magnitude higher than natural horseradish peroxidase (HRP), while catalase (CAT)-like and superoxide dismutase (SOD)-like activities of Ptc-PA are also comparable to those of natural enzymes. Biological experiments demonstrate that the detection limit of the Ptc-PA-based catalytic detection system exceeds that of visual inspection by 132-fold in clinical cancer diagnosis. Besides, Ptc-PA can reduce multi-organ acute inflammatory damage and mitigate oxidative stress disorder.

Mitochondria-targeting Cu2-xSe-TPP with dual enzyme activity alleviates Alzheimer's disease by modulating oxidative stress

Mitochondrial dysfunction in microglia has been implicated as a key pathogenesis of most neurodegenerative diseases including Alzheimer's disease (AD). Abnormal production of reactive oxygen species (ROS) and neuroinflammation caused by mitochondrial oxidative stress are important factors leading to neuronal death in AD. Herein, a "dual brake" strategy to synergistically halt mitochondrial dysfunction and neuroinflammation targeting mitochondria in microglia is proposed. To achieve this goal, (3-carboxypropyl) triphenyl-phosphonium bromide (TPP)-modified Cu2-xSe nanozymes (Cu2-xSe-TPP NPs) with dual enzyme-like activities was designed. Cu2-xSe-TPP NPs with superoxide dismutase-mimetic (SOD) and catalase-mimetic (CAT) activities can effectively scavenge ROS in the mitochondria of microglia and relieve mitochondrial oxidative stress. In vivo studies demonstrated that Cu2-xSe-TPP NPs can alleviate oxidative stress and promote neuroprotection in the hippocampus of AD model mice. In addition, Cu2-xSe-TPP NPs can regulate the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, promote Aβ phagocytosis and reshape the AD inflammatory microenvironment, thus effectively attenuating AD neuropathology and rescuing cognitive deficits in AD model mice. Taken together, this strategy preventing mitochondrial damage and remodeling the inflammatory microenvironment will provide a new perspective for AD therapy.

Highly sensitive colorimetric and paper-based detection for sildenafil in functional food based on monodispersed spherical magnetic graphene composite nanozyme

BackgroundSildenafil (SIL) is regarded as an illegal adulterant in functional foods. Some functional foods doped with SIL have posed significant concern about their safety risks. However, the facile colorimetric detection of SIL is rarely investigated. ResultsHerein, we prepared a monodispersed spherical composite nanozyme (Fe3O4–NH2/GONRs), possessing excellent peroxidase-like (POD-like) and catalase-like (CAT-like) activities and strong superparamagnetic property. The enzyme-like activities of Fe3O4–NH2/GONRs can be selectively inhibited by SIL due to the synergistic effect of hydrogen bonds and π-π stacking between Fe3O4–NH2/GONRs and SIL. Leveraging this mechanism, a highly sensitive and selective colorimetric detection for SIL with a detection limit (LOD) of 0.26 ng/mL was developed. In addition, we prepared a three-dimensional paper-based analytical device (3D-PAD) for SIL colorimetric detection with naked-eyes and the semi-quantitative analysis with a LOD of 88 ng/mL. SignificanceThe proposed colorimetric and PAD detections demonstrated the advantages of low-cost, highly sensitive and selective, thus have promise application potential in the rapid detection of adulterated functional foods.

Biocatalytic Clusterzyme Patches Restore Lung Function via Immunomodulation and Mitochondria Protection.

Currently, pulmonary complications such as lung infections during the perioperative period are still the main cause of prolonged hospitalization and death in patients with lung injury due to the lack of effective drugs. Clusterzyme, a kind of artificial enzyme with a high enzyme-like activity and safety profile, exhibits good effects on reducing oxidative stress and immunomodulation. Here, we present the functionalized patches that is administered on the lung airways and rescues the injured organ via clusterzymes. The long-term antioxidant capacity of the patches significantly ameliorated lipopolysaccharide-induced lung function impairment with a significant reduction in lung goblet cell metaplasia and oxidative stress. The inflammatory factors such as cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α levels decreased by 50%, while the mtDNA copy number increased by 50% and ATP production increased by 100%. Mice lung function was significantly improved, suggesting that the patches can rescue lung injury by modulating oxidative stress and immune responses as well as protecting the mitochondria, providing an avenue for effective intervention of lung injury.

Lattice expansion in ruthenium nanozymes improves catalytic activity and electro-responsiveness for boosting cancer therapy

Nanozymes have been attracting widespread interest for the past decade, especially in the field of cancer therapy, due to their intrinsic catalytic activities, strong stability, and ease of synthesis. However, enhancing their catalytic activity in the tumor microenvironment (TME) remains a major challenge. Herein, we manipulate catalytic activities of Ru nanozymes via modulating lattice spacing in Ru nanocrystals supported on nitrogen-doped carbon support, to achieve improvement in multiple enzyme-like activities that can form cascade catalytic reactions to boost cancer cell killing. In addition, the lattice expansion in Ru nanocrystals improve the responsiveness of the nanozymes to self-powered electric field, achieving maximized cancer therapeutic outcome. Under the electrical stimulation provided by a human self-propelled triboelectric device, the Ru-based nanozyme (Ru1000) with a lattice expansion of 5.99% realizes optimal catalytic performance and cancer therapeutic outcome of breast cancer in female tumor-bearing mice. Through theoretical calculations, we uncover that the lattice expansion and electrical stimulation promote the catalytic reaction, simultaneously, by reducing the electron density and shifting the d-band center of Ru active sites. This work provides opportunities for improving the development of nanozymes.

Enhanced peroxidase-like activity based on electron transfer between platinum nanoparticles and Ti3C2TX MXene nanoribbons coupled smartphone-assisted hydrogel platform for detecting mercury ions

BackgroundHeavy metal pollution poses a serious threat to the ecological environment. Mercury ion (Hg2+) is a class of highly toxic heavy metal ions, which is bioaccumulative, difficult to breakdown, and has a significant affinity with sulfur and thiol-containing proteins, which seriously affects environmental safety and human health. Nanozyme-based sensing methods are expected to be used to detect toxic heavy metal ions. However, the application of precious metal nanozymes to develop portable sensors with simplicity, high stability, and high sensitivity has not been explored to a large extent. ResultsIn this paper, based on MXene's unique adsorption capacity for certain precious metal ions, PtNPs/Ti3C2TXNR composites were successfully prepared by in-situ growth of Pt nanoparticles (PtNPs) on the surface of Ti3C2TX MXene nanoribbons (Ti3C2TXNR) using the hydrothermal technique. Experimental data revealed PtNPs/Ti3C2TXNR exhibited superior peroxidase-like activity, attributed to the synergistic effect of well-dispersed ultrasmall PtNPs and electron transfer effect. Hg2+ can significantly inhibit enzyme-like activity of PtNPs/Ti3C2TXNR due to specific capture and partial in-situ reduction of PtNPs, so a colorimetric sensor was constructed for ultra-trace detection of Hg2+ with a linear range of 0.2 nM and 400 nM. Furthermore, using the portable detecting capabilities of smartphones and hydrogel, a smartphone-assisted hydrogel sensing platform of Hg2+ was constructed. Notably, the two-mode sensing platforms exhibited outstanding detection performance with LOD values as low as 15 pM (colorimetric) and 26 pM (hydrogel), respectively, superior to recently reported nanozyme-based Hg2+ sensors. SignificanceCompared with other methods, the PtNPs/Ti3C2TXNR-based dual-mode sensor designed in this paper has superior sensitivity, high selectivity, simple operation and portability. In particular, the dual-output sensing strategy enables self-confirmation of detection results, greatly improving the reliability of the sensor, and is expected to be used for the on-site determination of trace mercury ions.

Breaking Barriers in Photothermal Tumor Therapy: A Cascade of Strain-Engineered Nanozyme in Action.

Cancer, a deadly and constantly evolving disease, has always been difficult to treat due to the complexity of the tumor microenvironment (TME). Cancer nanomedicines are proving to be a much better alternative for treatment due to their stability and ability to provide an efficient targeted therapy. An amorphous alloy bimetallene with an introduction of 2 % tensile strain with photothermal multiple enzyme-like catalytic activity is being presented here that functions as a TME-responsive nanozyme. Labeled as RhRu, this bimetallene, under acidic conditions, functions as oxidase (OXD) - like, peroxidase (POD) - like and catalase (CAT) - like enzymes, by producing radicals and disrupting the tumor cells. This effect is enhanced especially upon irradiation of laser and introduction of tensile strain in its heterophase boundaries. This current highlight discusses the strain engineering tactic of la-RhRu bimetallene and its potency as an anti-tumor therapeutic.

Nanozyme-Based Microfluidic Chip System for pH-Regulated Pretreatment and Sensitive Sensing.

Nanozymes, possessing nanomaterial properties and catalytic activities, offer great opportunities to design sensitive analytical detection systems. However, the low interference resistance of nanozymes poses a significant limitation on the precise detection of target substances. Herein, a nanozyme-based microfluidic chip system for pH-regulated pretreatment and sensitive sensing of cysteine (Cys) is reported. The copper metal-organic framework (Cu MOF) exhibits good cysteine oxidase-like activity at pH 7.0, while demonstrating excellent laccase-like activity at pH 8.0. Taking advantage of the pH-regulated enzyme-like activity, the integrated microfluidic device involving the immobilization of Cu MOF eliminates the interference of dopamine (DA) and accurately detects the target Cys. Compared with the untreated reaction system, the developed nanozyme system shows a significantly improved accuracy in detecting Cys, with an R2 value of 0.9914. This work provides an efficient method to enhance the interference resistance of nanozymes and broadens the application in sample pretreatment.

Biosynthesis of gold cluster nanozyme within the structure of TetX2 monooxygenase protein

The synthesis of gold nanoclusters within protein structures, such as TetX2 monooxygenase, presents a promising approach for obtaining nanostructures with enzyme-like activity. This is attributed to the presence of various amino acid groups that serve as biological templates for the formation and reduction of gold. The low toxicity, biocompatibility, solubility in aqueous systems, and specific fluorescence spectrum of these nanostructures enhance their utility in the detection of various analytes. The research involved expressing and purifying the recombinant enzyme TetX2, synthesizing gold on the TetX2 substrate, and investigating the effect of nanoclusters on the activity and structure of the TetX2 enzyme. The synthesis of gold nanoclusters(AuNCs) was carried out within the structure of TetX2. Finally, the accuracy of the AuNCs synthesis was verified by examining the morphology, size, spectroscopy, and catalytic activity. Results showed changes in the tertiary structure of the TetX2 enzyme in the presence of HAuCl4, loss of enzyme activity after the formation of gold nanoclusters, and successful incorporation of gold into the structure of TetX2. TetX2@AuNCs emitted blue light at 450 nm, and their catalytic properties were demonstrated through color changes in a chromogenic substrate (3,3′,5,5′-Tetramethylbenzidine-H2O2), indicating their authenticity. TetX2 is proposed as a new bio-template forthe synthesis of gold nanoclusters, with potential applications in biosensor design (Graphical abstract).

Hydrogel Microsphere-Encapsulated Bimetallic Nanozyme for Promoting Diabetic Bone Regeneration via Glucose Consumption and ROS Scavenging.

The healing of bone defects among diabetic patients presents a critical challenge due to the pathological microenvironment, characterized by hyperglycemia, excessive reactive oxygen species (ROS) production, and inflammation. Herein, multifunctional composite microspheres, termed GMAP are developed, using a microfluidic technique by incorporating Au@Pt nanoparticles (NPs) and GelMA hydrogel to modulate the diabetic microenvironment for promoting bone regeneration. The GMAP enables the sustained release of Au@Pt NPs, which function as bimetallic nanozymes with dual enzyme-like activities involving glucose oxidase and catalase. The synergistic effect allows for efficient glucose consumption and ROS elimination concurrently. Thus, the GMAP effectively protects the proliferation of bone marrow mesenchymal stem cells (BMSCs) under adverse high-glucose conditions. Furthermore, it also promotes the osteogenic differentiation and paracrine capabilities of BMSCs, and subsequently inhibits inflammation and enhances angiogenesis. In vivo diabetic rats bone defect model, it is demonstrated that GMAP microspheres significantly improve bone regeneration, as verified by micro-computed tomography and histological examinations. This study provides a novel strategy for bone regeneration by modulating the diabetic microenvironment, presenting a promising approach for addressing the complex challenges associated with bone healing in diabetic patients.