Glutathione Peroxidase-like Activity Research Articles

Shell-core COF@Co3O4 Z-scheme heterojunctions for triple amplification of oxidative stress to enhance nanocatalytic-sonodynamic tumor therapy

Constructing efficient nanozyme platforms with multiple amplification of tumor oxidative stress is crucial to enhance the reactive oxygen species (ROS)-mediated tumor therapy. However, the inherent low catalytic activity and unsustainability of nanozymes in the highly complex tumor microenvironment (TME) severely restricted their clinical applications. Herein, we first reported a heterojunction (HJ)-enhanced nanocatalytic-sonodynamic therapy nanoplatform based on COF@Co3O4 Z-scheme HJs with shell-core architecture by coating COF on the surface of Co3O4 nanospheres. The as-prepared Co3O4 nanospheres not only exhibited excellent sonodynamic properties owing to the narrow bandgap (1.37 eV), but also possessed peroxidase-like, catalase-like, and glutathione peroxidase-like catalytic activities to realize the amplification of ROS levels and relieve tumor hypoxia due to the presence of multivalent Co element. More importantly, the ultrasound (US)-triggered ROS generation ability and triple enzyme-mimic activity of Co3O4 nanospheres were greatly enhanced by the encapsulation of COF to fabricate Z-scheme HJs with large interfacial contact area. Based on the improved spatial separation dynamics of US-generated electron-hole pairs and accelerate carrier transfer process, complete tumor eradication without recurrence was realized by the synergetic therapeutic effect of COF@Co3O4 through HJ-enhanced nanocatalytic-sonodynamic therapy. This work presents highly efficient nanozyme platforms with both multiple enzyme-mimic catalytic activity and sonodynamic properties, which will open up a promising approach to engineer semiconductor Z-scheme HJs for enhanced tumor therapy.

3,3'-Diselenodipropionic acid immobilised gelatin gel: a biomimic catalytic nitric oxide generating material for topical wound healing application.

Nitric oxide (NO) plays a pivotal role in the wound healing process and promotes the generation of healthy endothelium. In this work, a simple method has been developed for fabricating a diselenide grafted gelatin gel, which reduces NO donors such as S-nitroso-N-acetylpenicillamine (SNAP) by glutathione peroxidase-like mechanism to produce NO. Briefly, the process involved covalently conjugating 3,3'-diselenodipropionic acid (DSePA) with gelatin via carbodiimide coupling. The resulting gelatin-DSePA conjugate (G-Se-Se-G) demonstrated NO production upon incubation with SNAP and glutathione (GSH) with the flux of 4.8 ± 0.6 nmol cm-2 min-1 and 1.6 ± 0.1 nmol cm-2 min-1 at 10 min and 40 min, respectively. The G-Se-Se-G recovered even after 5 days of incubation with the reaction mixture retaining catalytic activity up to 74%. Subsequently, G-Se-Se-G was suspended (5% w/v) in water with lecithin (6% w/w of gelatin) and F127 (3% w/w of gelatin) to prepare gel through temperature dependant gelation method. The fabricated G-Se-Se-G gel exhibited desirable rheological characteristics and excellent mechanical stability under storage conditions and did not cause any significant toxicity in normal human keratinocytes (HaCaT) and fibroblast cells (WI38) up to 50 μg ml-1 of selenium equivalent. Finally, mice studies confirmed that topically applied G-Se-Se-G gel and SNAP promoted faster epithelization and collagen deposition at the wound site. In conclusion, the development of a biomimetic NO generating gel with sustained activity and biocompatibility was achieved.

Fe/Cu-AuNP nanocomposites as enzyme-like catalysts to modulate the tumor microenvironment for enhanced synergistic cancer therapy.

The intensive investigation of chemodynamic therapy (CDT) for tumor eradication revealed that the therapeutic effects of this ROS-mediated therapy are limited by endogenous reductants and inefficient Fenton-like reactions. In this study, we developed a new Fe/Cu-AuNP-PEG nanocomposite to enhance CDT and provide a synergistic treatment for tumors. The Fe/Cu-AuNP-PEG nanocomposite demonstrated effective ˙OH production and high photothermal conversion efficiency under 808 nm illumination, which promoted the ˙OH production, thereby enhancing the CDT efficacy and exhibiting a synergistic treatment for cancer. More importantly, the Fe/Cu-AuNP-PEG nanocomposite showed the ability to deplete GSH and catalyze glucose to generate H2O2, which facilitated the Fenton-like reaction and reduced the antioxidant properties of tumors, further improving the efficacy of CDT. Therefore, the Fe/Cu-AuNP-PEG nanocomposite, with horseradish peroxidase-like, glutathione peroxidase-like, and glucose oxidase-like activities, is a promising anti-tumor agent for integrating enhanced CDT and photothermal therapy (PTT) with the enhancement of synergistic therapeutic effects.

Multifunctional tadpole-like bimetallic nanoparticles realizes synergistic sterilization with chemical kinetics and photothermal therapy

Bacterial infection has become a global health issue. The misuse of antibiotics has been resulting in increased drug resistance and bioaccumulation. Therefore, developing a highly safe antibacterial agent, with high antibacterial performance is demanding. Inspired by the natural motors performing automated tasks in complicated living environments, we demonstrate tadpole-like nanoparticles (TNPs) with several functions, including high photothermal conversion efficiency, peroxidase-like catalytic activity, glutathione peroxidase-like activity, and catalase-like activity. TNPs produce hydroxyl radical (•OH) at an extremely low concentration of hydrogen peroxide of 0.006%, which can damage bacterial cell membranes, proteins, and DNA. Moreover, the glutathione peroxidase-like activity disrupts the anti-oxidative mechanism of bacteria and improves the permeability of the cell membranes, consequently enhancing the killing effect of ROS. In addition, TNPs possess tadpole-like asymmetry to overcome Brownian motion, demonstrating strong directional motion propelled by O2. The in vivo experiments indicate that TNPs could also shorten the inflammatory period and promote angiogenesis, making them a very promising antibacterial agent.

Click Chemistry of Selenium Dihalides: Novel Bicyclic Organoselenium Compounds Based on Selenenylation/Bis-Functionalization Reactions and Evaluation of Glutathione Peroxidase-like Activity

A number of highly efficient methods for the preparation of novel derivatives of 9-selenabicyclo[3.3.1]nonane in high yields based on selenium dibromide and cis,cis-1,5-cyclooctadiene are reported. The one-pot syntheses of 2,6-diorganyloxy-9-selenabicyclo[3.3.1]nonanes using various O-nucleophiles including alkanols, phenols, benzyl, allyl, and propargyl alcohols were developed. New 2,6-bis(1,2,3-triazol-1-yl)-9-selenabicyclo[3.3.1]nonanes were obtained by the copper-catalyzed 1,3-dipolar cycloaddition of 2,6-diazido-9-selenabicyclo[3.3.1]nonane with unsubstituted gaseous acetylene and propargyl alcohol. The synthesis of 2,6-bis(vinylsulfanyl)-9-selenabicyclo[3.3.1]nonane, based on the generation of corresponding dithiolate anion from bis[amino(iminio)methylsulfanyl]-9-selenabicyclo[3.3.1]nonane dibromide, followed by the nucleophilic addition of the dithiolate anion to unsubstituted acetylene, was developed. The glutathione peroxidase-like activity of the obtained water-soluble products was estimated and compounds with high activity were found. Overall, 2,6-Diazido-9-selenabicyclo[3.3.1]nonane exhibits the highest activity among the obtained compounds.

Single-atom nanozymes catalytically surpassing naturally occurring enzymes as sustained stitching for brain trauma

Regenerable nanozymes with high catalytic stability and sustainability are promising substitutes for naturally-occurring enzymes but are limited by insufficient and non-selective catalytic activities. Herein, we developed single-atom nanozymes of RhN4, VN4, and Fe-Cu-N6 with catalytic activities surpassing natural enzymes. Notably, Rh/VN4 preferably forms an Rh/V-O-N4 active center to decrease reaction energy barriers and mediates a “two-sided oxygen-linked” reaction path, showing 4 and 5-fold higher affinities in peroxidase-like activity than the FeN4 and natural horseradish peroxidase. Furthermore, RhN4 presents a 20-fold improved affinity in the catalase-like activity compared to the natural catalase; Fe-Cu-N6 displays selectivity towards the superoxide dismutase-like activity; VN4 favors a 7-fold higher glutathione peroxidase-like activity than the natural glutathione peroxidase. Bioactive sutures with Rh/VN4 show recyclable catalytic features without apparent decay in 1 month and accelerate the scalp healing from brain trauma by promoting the vascular endothelial growth factor, regulating the immune cells like macrophages, and diminishing inflammation.

Nitric oxide-mediated regulation of mitochondrial protective autophagy for enhanced chemodynamic therapy based on mesoporous Mo-doped Cu9S5 nanozymes

The depletion of reactive oxygen species (ROS) by glutathione (GSH) and oxidative stress induced protective autophagy severely impaired the therapeutic effect of chemodynamic therapy (CDT). Therefore, how to construct a CDT treatment nanosystem with high yield and full utilization of ROS in tumor site is the main issue of CDT. Herein, a multifunctional cascade bioreactor based on mesoporous Mo-doped Cu9S5 (m-MCS) nanozymes loaded with L-Arginine (LA), abbreviated as m-MCS@LA, is constructed for realizing enhanced CDT promoted by ultrasound (US) triggered gas therapy. The m-MCS based on the catalytic performance of multivalent metal ions, which were served as nanozymes, exhibit enhanced Fenton-like and glutathione (GSH) peroxidase-like activities in comparison to Cu9S5 nanoparticles without Mo-doping. Once placed in tumor microenvironment (TME), the existence of redox couples (Cu+/Cu2+ and Mo4+/Mo6+) in m-MCS enabled it to react with hydrogen peroxide (H2O2) to generate ·OH for achieving CDT effect via Fenton-like reaction. Meanwhile, m-MCS could consume overexpressed GSH in tumor microenvironment (TME) to alleviate antioxidant capability for enhancing CDT effect. Moreover, m-MCS with mesoporous structure could be employed as the carrier to load natural nitric oxide (NO) donor LA. US as the excitation source with high tissue penetration can trigger m-MCS@LA to produce NO. As the gas transmitter with physiological functions, NO could play dual roles to kill cancer cells through gas therapy directly, and enhance CDT effect by inhibiting protective autophagy simultaneously. As a result, this US-triggered and NO-mediated synergetic cancer chemodynamic/gas therapy based on m-MCS@LA NPs can effectively eliminate primary tumor and achieved tumor-specific treatment, which provide a possible strategy for developing more effective CDT in future practical applications. Statement of SignificanceThe depletion of reactive oxygen species (ROS) by glutathione (GSH) and oxidative stress induced protective autophagy severely impaired the therapeutic effect of chemodynamic therapy (CDT). Herein, a multifunctional cascade bioreactor based on mesoporous Mo-doped Cu9S5 (m-MCS) nanozymes loaded with L-Arginine (m-MCS@LA) is constructed for realizing enhanced CDT promoted by ultrasound (US) triggered gas therapy. The m-MCS with double redox couples presents the enhanced enzyme-like activities to perform cascade reactions for reducing GSH and generating ROS. LA loaded by m-MCS can produce NO triggered by US to inhibit the mitochondria protective autophagy for reactivating mitochondria involved apoptosis pathway. The US-triggered and NO-mediated CDT based on m-MCS@LA can effectively eliminate primary tumor through the high yield and full utilization of ROS.

Inhibition of H1N1 Influenza Virus-induced Apoptosis by Ebselen Through ROS-mediated ATM/ATR Signaling Pathways.

Influenza A viruses can cause global outbreaks and seasonal pandemics. However, the use of conventional anti-influenza drugs leads to an increase in drug-resistant mutations in influenza viruses worldwide. Therefore, numerous studies have focused on developing effective anti-influenza drugs. It is feasible to treat influenza by targeting influenza-mediated oxidative damage. Ebselen is a synthetic organoselenium compound which provides glutathione peroxidase-like activity. It has been shown to play a role in anti-influenza therapy, but the mechanism remains to be further explored. This experiment verified the anti-influenza effect of ebselen. CCK-8 and PCR showed that ebselen had a significant inhibitory effect on virus replication compared with the virus group. In addition, the mechanistic investigations revealed that ebselen could inhibit influenza-mediated apoptosis, mitochondrial damage, accumulation of reactive oxygen species, and DNA breakage. At the same time, ebselen significantly inhibited the phosphorylation of ATM and ATR and promoted the activation of PARP and Caspase-3. Ebselen, on the other hand, reduced the inflammatory response caused by influenza. These results suggest that ebselen is a promising inhibitor for H1N1.

Regio- and Stereoselective Synthesis of (Z,Z)-Bis(3-amino-3-oxo-1-propenyl) Selenides and Diselenides Based on 2-propynamides: A Novel Family of Diselenides with High Glutathione Peroxidase-like Activity

The efficient regio- and stereoselective syntheses of (Z,Z)-bis(3-amino-3-oxo-1-propenyl) selenides and diselenides in high yields based on the nucleophilic addition of sodium selenide to 2-propynamides and sodium diselenide to 3-(trimethylsilyl)-2-propynamides have been developed. The first examples of the addition of a selenium-centered nucleophile to 2-propynamides with a terminal triple bond and diselenide anion to 3-(trimethylsilyl)-2-propynamides have been carried out. Bis(3-amino-3-oxo-1-propenyl) diselenides are a novel family of compounds, none of which has yet been described in the literature. The glutathione peroxidase-like activity of the obtained compounds has been evaluated and products with high activity have been found. It was established that diselenides are superior to selenides with the same substituents in glutathione peroxidase-like activity. The results of the structural studying of products by single-crystal X-ray diffraction analysis and 77Se-NMR data are discussed.

Cold stratification-induced dormancy removal in apple (Malus domestica Borkh.) seeds is accompanied by an increased glutathione pool in embryonic axes

A reduced form of glutathione (GSH) is an essential metabolite that participates in the control of reactive oxygen species (ROS) levels in cells. GSH plays a pivotal role in seed biology as a modulator of seed viability and germination. The GSH:GSSG ratio and half-cell reduction potential (EGSSG/2GSH) serve as indicators of the oxidative status in seeds. Apple (Malus domestica Borkh.) seeds are deeply dormant, and this state is removed by long-term cold stratification. The aim of our work was to examine the modification of GSH and GSSG content, GSH:GSSG ratio and EGSSG/2GSH in the embryonic axes isolated from apple seeds subjected to cold stratification for 7, 14, 21 and 40 d. Our data indicated that cold stratification increased the generation of free radicals in the embryonic axes, which correlated with an alteration in the expression of genes encoding Rboh, particularly RbohC. GSH and GSSG levels increased during prolonged cold stratification of apple seeds. This was accompanied by the modification of glutathione reductase and glutathione peroxidase-like activities, which did not match their transcript levels. The steady-state GSH:GSSG ratio and EGSSG/2GSH in the axes of embryos subjected to cold stratification indicated no impact of the dormancy removal treatment on apple seed viability. We suggest that the glutathione system is an important component of the redox network and is involved in the management of the seed transition from dormant to nondormant states.

Biomimetic Nanocatalysts Enhance Oxidative Damage against Cancer Cells

Red blood cells (RBCs) naturally delivery oxygen and kill pathogens through reactive oxygen species (ROS) generated from Fe-protoporphyrin component in hemoglobin, which might be also promising for cancer treatment in terms of relieving tumor hypoxia and inducing oxidative damage against cancer cells. However, the therapy efficacy is far from satisfactory due to the limited oxygen transport and ROS generation rate in tumor tissue. Herein, artificial RBCs (designated as FTP@RBCM) with radical storm production ability is developed for oncotherapy through multi-dimensional reactivity pathways of Fe-protoporphyrin based hybrid metal-organic framework (FTP, as the core), including photodynamic/chemodynamic (PDT/CDT)-like, catalase-like and glutathione (GSH) peroxidase-like activity. Meanwhile, owing to the advantages of reticuloendothelial system (RES) evasion and long circulation abilities of RBCs governed by their cell membranes (RBCMs), FTP with surface further coated with RBCMs (FTP@RBCM) could enormously accumulate at tumor site to achieve remarkably enhanced therapy efficiency.

Surface engineered iron oxide nanozyme for synergistic chemodynamic/photodynamic therapy with glutathione depletion and hypoxia relief

Reactive oxygen species (ROS)-based therapies, such as photodynamic therapy (PDT) and chemodynamic therapy (CDT), hold great promise to cancer treatment. However, their efficacies are subject to hypoxia and overexpressed glutathione (GSH) in tumor microenvironment (TME). Herein, we develop Mn (II) ions and pyropheophorbide (PPa) engineered iron oxide nanoparticles (IMOP) to regulate tumor hypoxia and deplete GSH for synergistic CDT/PDT. In the TME, IMOP could catalytically decompose hydrogen peroxide (H2O2) to hydroxyl radicals (·OH) as a CDT agent. Significantly, IMOP exhibits high catalase-like and glutathione peroxidase-like activities simultaneously. Once enriching into tumor, IMOP could catalyze H2O2 to oxygen (O2) to overcome hypoxic TME for improved 1O2 generation via PPa-mediated PDT. Meanwhile, the glutathione peroxidase-like activity ensure IMO to reduce the intratumoral GSH concentration and further reduce the consumption of active ROS during therapy. We demonstrated that the regulation by IMOP elicited synergistic CDT/PDT efficacy for enhanced ROS-based cancer treatment both in vitro and in vivo. Moreover, the existence of Mn (II) allowed IMO with T1 contrast behavior to monitor the progress of CDT/PDT in magnetic resonance imaging. This work provides a new guidance for designing TME-based anticancer platform for effective and precise cancer treatment.

Red Blood Cell-Mimic Nanocatalyst Triggering Radical Storm to Augment Cancer Immunotherapy

HighlightsA red blood cell-mimic nanocatalyst with photodynamic/chemodynamic-like, catalase-like and glutathione peroxidase-like activities was developed to boost radical storms for tumor eradication.Combined with the Tim-3 immune checkpoint blockade, such radical therapy can systematically evoke a robust systemic antitumor immune response to eliminate residual cancer cells.

Selenium Dihalides Click Chemistry: Highly Efficient Stereoselective Addition to Alkynes and Evaluation of Glutathione Peroxidase-Like Activity of Bis(E-2-halovinyl) Selenides.

Highly efficient stereoselective syntheses of novel bis(E-2-chlorovinyl) selenides and bis(E-2-bromovinyl) selenides in quantitative yields by reactions of selenium dichloride and dibromide with alkynes were developed. The reactions proceeded at room temperature as anti-addition giving products exclusively with (E)-stereochemistry. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. The influence of substituents in the products on their glutathione peroxidase-like activity was discussed.

Atomic Engineering of Clusterzyme for Relieving Acute Neuroinflammation through Lattice Expansion.

Natural enzymes are efficient and versatile biocatalysts but suffer in their environmental tolerance and catalytic stability. As artificial enzymes, nanozymes can improve the catalytic stability, but it is still a challenge to achieve high catalytic activity. Here, we employed atomic engineering to build the artificial enzyme named Au24Ag1 clusterzyme that hosts an ultrahigh catalytic activity as well as strong physiological stability via atom manipulation. The designed Au24Ag1 clusterzyme activates the Ag-S active site via lattice expansion in the oligomer atom layer, showing an antioxidant property 72 times higher than that of natural antioxidant Trolox. Enzyme-mimicked studies find that Au24Ag1 clusterzyme exhibits high catalase-like (CAT-like) and glutathione peroxidase-like (GPx-like) activity with a maximum reaction rate of 68.9 and 17.8 μM/min, respectively. Meanwhile, the unique catalytic landscape exhibits distinctive reactions against inflammation by inhibiting the cytokines at an early stage in the brain. Atomic engineering of clusterzymes provides a powerful and attractive platform with satisfactory atomic dispersion for tailoring biocatalysts freely at the atomic level.

Catalytically potent and selective clusterzymes for modulation of neuroinflammation through single-atom substitutions

Emerging artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have long been pursued with sustained efforts. The majority of current candidates have rather poor catalytic activity compared with natural molecules. To tackle this limitation, we design artificial enzymes based on a structurally well-defined Au25 cluster, namely clusterzymes, which are endowed with intrinsic high catalytic activity and selectivity driven by single-atom substitutions with modulated bond lengths. Au24Cu1 and Au24Cd1 clusterzymes exhibit 137 and 160 times higher antioxidant capacities than natural trolox, respectively. Meanwhile, the clusterzymes demonstrate preferential enzyme-mimicking catalytic activities, with Au25, Au24Cu1 and Au24Cd1 displaying compelling selectivity in glutathione peroxidase-like (GPx-like), catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) activities, respectively. Au24Cu1 decreases peroxide in injured brain via catalytic reactions, while Au24Cd1 preferentially uses superoxide and nitrogenous signal molecules as substrates, and significantly decreases inflammation factors, indicative of an important role in mitigating neuroinflammation.

(Z,Z)-Selanediylbis(2-propenamides): Novel Class of Organoselenium Compounds with High Glutathione Peroxidase-Like Activity. Regio- and Stereoselective Reaction of Sodium Selenide with 3-Trimethylsilyl-2-propynamides.

The efficient regio- and stereoselective synthesis of (Z,Z)-3,3′-selanediylbis(2-propenamides) in 76–93% yields was developed based on the reaction of sodium selenide with 3-trimethylsilyl-2-propynamides. (Z,Z)-3,3′-Selanediylbis(2-propenamides) are a novel class of organoselenium compounds. To date, not a single representative of 3,3′-selanediylbis(2-propenamides) has been described in the literature. Studying glutathione peroxidase-like properties by a model reaction showed that the activity of the obtained products significantly varies depending on the organic moieties in the amide group. Divinyl selenide, which contains two lipophilic cyclohexyl substituents in the amide group, exhibits very high glutathione peroxidase-like activity and this compound is considerably superior to other products in this respect.

Mechanisms of ebselen as a therapeutic and its pharmacology applications.

Ebselen is a synthetic organoselenium radical scavenger compound that possesses glutathione peroxidase-like activity and its own unique bioactivity by reacting with thiols, hydroperoxides and peroxynitrites. Owing to its high affinity towardseveral essential reactions, ebselen protects cellular components from oxidative and free radical damage, and it has been employed as a useful tool for studying redox-related mechanisms. Based on numerous in vitro and in vivo research, mechanisms are proposed to understand the biomedical and molecular actions of ebselen in health and disease, and it is currently under clinical trials for the prevention and treatment of various human disorders. Based on these outstanding discoveries, this review summarizes the current understanding of the biochemical and molecular characteristics, pharmacological applications and future directions of ebselen.

New insight into the role of glutathione reductase in glutathione peroxidase-like activity determination by coupled reductase assay: Molecular Docking Study

Previously we have shown that among 15 substituted salicyloyl (2-hydroxybenzoyl) 5-seleninic acids (SSAs) 4 compounds with longer side chains or a cyclohexyl group exhibit no glutathione peroxidase (GPx)-like activity in the coupled reductase assay. Experimental inhibition of glutathione reductase (GR) by the selenenylsulfide (a main intermediate in the catalytic cycle for GPx-like activity determination) of one of the inactive compounds led us to assess the interactions between 15 selenenylsulfide compounds and the active site of GR by molecular docking. Docking results showed that S and Se atoms in selenenylsulfides of the compounds with no GPx-like activity were beyond 5 Å from S atom of Cys-58 or N atom of imidazole ring of His-467 (Root Mean Square Distances for general assessment of 3 major distances were over 4.8 Å) in the active site, so that they could not be catalyzed to be reduced by GR. Furthermore, their docking scores over 89 Kcal/mol meant that the selenenylsulfides were bound too strongly to the active site to leave it, leading eventually to inhibition of GR. We also applied the molecular docking to other GPx mimics such as ebselen, cyclic seleninate esters and di(propylaminomethylphenyl) diselenides to explain the differences in their GPx-like activity depending to the assays used. Our results suggest that the reduction of a selenenylsulfide by GR plays a positive role in GPx-like activity of GPx mimics in the coupled assay and recommended the prediction of possibility and strength of GPx-like activity by molecular docking before entering experimental research.

ZIF-67 as a Template Generating and Tuning "Raisin Pudding"-Type Nanozymes with Multiple Enzyme-like Activities: Toward Online Electrochemical Detection of 3,4-Dihydroxyphenylacetic Acid in Living Brains.

Due to its unique structure and high porosity, metal-organic frameworks (MOFs) can act not only as nanozyme materials but also as carriers to encapsulate natural enzymes and thus have received extensive attention in recent years. However, a few research studies have been conducted to investigate MOF as a template to generate and tune nanozymes in the structure and performance. In this work, the "raisin pudding"-type ZIF-67/Cu0.76Co2.24O4 nanospheres (ZIF-67/Cu0.76Co2.24O4 NSs) were obtained by rationally regulating the weight ratio of ZIF-67 and Cu(NO3)2 in the synthesis process. Here, ZIF-67 not only acts as a template but also provides a cobalt source for the synthesis of cobalt copper oxide on the surface of ZIF-67/Cu0.76Co2.24O4 NSs with multiple enzyme-like activities. The ZIF-67/Cu0.76Co2.24O4 NSs can mimic four kinds of enzymes with peroxidase-like, glutathione peroxidase-like, superoxide dismutase-like, and laccase-like activities. Based on its laccase-like activity, an online electrochemical system for continuous monitoring of 3,4-dihydroxyphenylacetic acid with good linearity in the range of 0.5-20 μM and a detection limit of 0.15 μM was established. Furthermore, the alteration of DOPAC in the brain microdialysate before and after ischemia of the rats' brain was also successfully recorded. This work not only raises a new idea for the synthesis of nanozyme materials with multiple enzyme activities but also provides a new solution for the detection of neurotransmitters in living brains.