Inorganic Nanozymes Research Articles

Cobalt (II) porphyrin nanoaggregates as sacrificial templates to improve the peroxidase-like activity of light-controlled TiO2-based nanozymes for colorimetric determination of amikacin

Although porphyrin modification can improve the peroxidase-like activity of some inorganic nanozymes, it is hardly studied that metal porphyrin self-assembled nanoaggregates as sacrificial templates to turn on the peroxidase-like activity of inorganic nanozymes under light illumination. In this work, cobalt (II) 5,10,15,20-Tetrakis (4-carboxylpheyl)porphyrin (CoTCPP) self-assembled nanoaggregates are firstly used as soft templates to prepare TiO2-based nanozymes with the enhanced peroxidase-like activity. Interestingly, CoTCPP nanoaggregates can be changed into Co oxide nanoparticles dispersed into the nanosphere composites. Furthermore, the peroxidase-like activity of CoTCPP-TiO2 nanospheres can be controlled by light illumination. Comparatively, CoTCPP-TiO2 nanoshperes exhibit the highest peroxidase-like activity of three nanospheres (CoTCPP-TiO2, H2TCPP-TiO2 and TiO2) with similar morphology under the light illumination. Other than the existence of oxygen vacancy, the formation of heterostructure between TiO2 and a small amount of Co3O4 are ascribed to increase the catalytic activity of CoTCPP-TiO2 composites. Thus, a facile and convenient colorimetric sensing platform has been constructed and tuned by light illumination for determining H2O2 and amikacin in a good linear range of 20–100 and 50–100 μM with a limit of detection (LOD) of 3.04 μM and 1.88 μM, respectively. The CoTCPP-TiO2 based colorimetric sensing platform has been validated by measuring the amikacin residue in lake water.

AI-Powered Knowledge Base Enables Transparent Prediction of Nanozyme Multiple Catalytic Activity.

Nanozymes are unique materials with many valuable properties for applications in biomedicine, biosensing, environmental monitoring, and beyond. In this work, we developed a machine learning (ML) approach to search for new nanozymes and deployed a web platform, DiZyme, featuring a state-of-the-art database of nanozymes containing 1210 experimental samples, catalytic activity prediction, and DiZyme Assistant interface powered by a large language model (LLM). For the first time, we enable the prediction of multiple catalytic activities of nanozymes by training an ensemble learning algorithm achieving R2 = 0.75 for the Michaelis-Menten constant and R2 = 0.77 for the maximum velocity on unseen test data. We envision an accurate prediction of multiple catalytic activities (peroxidase, oxidase, and catalase) promoting novel applications for a wide range of surface-modified inorganic nanozymes. The DiZyme Assistant based on the ChatGPT model provides users with supporting information on experimental samples, such as synthesis procedures, measurement protocols, etc. DiZyme (dizyme.aicidlab.itmo.ru) is now openly available worldwide.

Engineering inorganic nanozyme architectures for decomposition of reactive oxygen species.

Enzyme-mimicking nanomaterials (nanozymes) with antioxidant activity are at the forefront of research efforts towards biomedical and industrial applications. The selection of enzymatically active substances and their incorporation into novel inorganic nanozyme structures is critically important for this field of research. To this end, the fabrication of composites can be desirable as these can either exhibit multiple enzyme-like activities in a single material or show increased activity compared to the nanozyme components. Conversely, by modifying the structure of a nanomaterial, enzyme-like activities can be induced in formerly inert particles. We identify herein the three main routes of composite nanozyme synthesis, namely, surface functionalization of a particle with another compound, heteroaggregation of individual nanozymes, and modification of the bulk nanozyme structure to achieve optimal antioxidant activity. We discuss in particular the different inorganic support materials used in the synthesis of nanozyme architectures and the advantages brought forth by the use of composites.

Peroxidase-like Activity of CeO2 Nanozymes: Particle Size and Chemical Environment Matter.

The enzyme-like activity of metal oxide nanoparticles is governed by a number of factors, including their size, shape, surface chemistry and substrate affinity. For CeO2 nanoparticles, one of the most prominent inorganic nanozymes that have diverse enzymatic activities, the size effect remains poorly understood. The low-temperature hydrothermal treatment of ceric ammonium nitrate aqueous solutions made it possible to obtain CeO2 aqueous sols with different particle sizes (2.5, 2.8, 3.9 and 5.1 nm). The peroxidase-like activity of ceria nanoparticles was assessed using the chemiluminescent method in different biologically relevant buffer solutions with an identical pH value (phosphate buffer and Tris-HCl buffer, pH of 7.4). In the phosphate buffer, doubling CeO2 nanoparticles' size resulted in a two-fold increase in their peroxidase-like activity. The opposite effect was observed for the enzymatic activity of CeO2 nanoparticles in the phosphate-free Tris-HCl buffer. The possible reasons for the differences in CeO2 enzyme-like activity are discussed.

Integrated cascade catalysis of microalgal bioenzyme and inorganic nanozyme for anti-inflammation therapy.

Combinations of multiple enzymes for cascade catalysis have been widely applied in biomedicine, but the integration of a natural bioenzyme with an inorganic nanozyme is less developed. Inspired by the abundant content of superoxide dismutase (SOD) in Spirulina platensis (SP), we establish an integrated cascade catalysis for anti-inflammation therapy by decorating catalase (CAT)-biomimetic ceria nanoparticles (CeO2) onto the SP surface via electrostatic interaction to build microalgae-based biohybrids. The biohybrids exhibit combined catalytical competence for preferentially transforming superoxide anion radicals (O2˙-) to hydrogen peroxide (H2O2), and subsequently catalyzing H2O2 disproportionation to water and oxygen. In ulcerative colitis and Crohn's disease, the biohybrids reveal a satisfactory therapeutic effect owing to the synergistic reactive oxygen species (ROS)-scavenging capacity, suggesting a new train of thought for enzyme-based biomedical application.

Copper-based inorganic nanozymes enhance the electrical conductivity of tumors to synergistically induce the pyroptosis, ferroptosis, and apoptosis of tumors

Electrotherapy (ET) effectively ablates solid tumors, inhibiting their growth.

SnSe Nanosheets Mimic Lactate Dehydrogenase to Reverse Tumor Acid Microenvironment Metabolism for Enhancement of Tumor Therapy.

The acidic tumor microenvironment (TME) is unfriendly to the activity and function of immune cells in the TME. Here, we report inorganic nanozymes (i.e., SnSe NSs) that mimic the catalytic activity of lactate dehydrogenase to degrade lactate to pyruvate, contributing to the metabolic treatment of tumors. As found in this study, SnSe NSs successfully decreased lactate levels in cells and tumors, as well as reduced tumor acidity. This is associated with activation of the immune response of T cells, thus alleviating the immunosuppressive environment of the TME. More importantly, the nanozyme successfully inhibited tumor growth in mutilate mouse tumor models. Thus, SnSe NSs show a promising result in lactate depletion and tumor suppression, which exemplifies its potential strategy in targeting lactate for metabolic therapy.

Intrinsic Light-Activated Oxidase Mimicking Activity of Conductive Polyaniline Nanofibers: A Class of Metal-Free Nanozyme.

In recent decades, studies have focused on inorganic nanozymes to overcome the intrinsic drawbacks of bioenzymes due to the demands of improving the reaction conditions and lack of robustness to harsh environmental factors. Many biochemical reactions catalyzed by enzymes require light activation. Light-activated nanozymes have distinct advantages, including being regulated by light stimuli, activating the molecular oxygen to produce reactive oxygen species (ROS) without interfering supplementary oxidants, and often showing a synergistic effect to catalyze some challenging reactions. Only a few studies have been done on this connection. Therefore, it is still a big challenge to develop a nanozyme regulated by light activation. Herein, we uncovered the light-activated oxidase mimicking activity of a conducting polymer polyaniline nanofibers (PANI-NFs). PANI-NFs exhibit intrinsic light-activated brilliant oxidase-like activity, can catalyze the colorless tetramethyl benzidine (TMB) to produce a blue product TMBox, and have a distinct Km = 0.087 mM and a high Vmax = 2.32 μM min-1 value, measured by using Hanes-Woolf kinetics. We also report the light-activated oxidase activity of some other renowned carbocatalysts graphene oxide and graphitic carbon nitride and compare them with PANI-NFs. This type of property shown by the conductive polymer is amazing. The density functional theory is used to verify the stability and the mode of adsorption of the PANI NFs-TMB composite, which corroborates the experimental results. Furthermore, the current nanozyme demonstrated a significant ability to kill both Gram-negative and Gram-positive bacteria as well as effectively destroy biofilms under physiological conditions. We believe that this work provides the motivation to create a link between optoelectronics and biological activity in the near future.

Multi-functional oxidase-like activity of praseodymia nanorods and nanoparticles

The ability to mimic protein-based oxidase with multi-functional inorganic nanozymes would greatly advance biomedical and clinical practices. Praseodymia (PrOx) nanorods (NRs) and nanoparticles (NPs) have been synthesized using hydrothermal and precipitation methods. Both PrOx catalysts with different morphologies exhibit significantly higher oxidase-like activities (Michaelis-Menten constant Km ≤ 0.026 mM) than commercial PrOx and most so-far-reported artificial enzymes. One of the substrates, dopamine, can be oxidized and further polymerized to generate polydopamine in acidic conditions. Akin to CeO2, which is a well-studied nanozyme, a different mechanism involving holes+, oxygen vacancies and oxygen mobility over PrOx catalysts has been proposed in this work. However, fluoride ions were found to impose opposite effects on the oxidase-mimicking activity of PrOx and CeO2, implying a promising path for the exploration of new nanozymes. In support of this, PrOx was further applied in colorimetric sensing of L-cysteine and fluoride with high sensitivity.

DiZyme: Open-Access Expandable Resource for Quantitative Prediction of Nanozyme Catalytic Activity.

Enzymes suffer from high cost, complex purification, and low stability. Development of low-cost artificial enzymes of comparative or higher effectiveness is desired. Given its complexity, it is desired to presume their activities prior to experiments. While computational approaches demonstrate success in modeling nanozyme activities, they require assumptions about the system to be made. Machine learning (ML) is an alternative approach towards data-driven material property prediction achieving high performance even on multicomponent complex systems. Despite the growing demand for customized nanozymes, there is no open access nanozyme database. Here, a user-friendly expandable database of >300 existing inorganic nanozymes is developed by data collection from >100 articles. Data analysis is performed to reveal the features responsible for catalytic activities of nanozymes, and new descriptors are proposed for its ML-assisted prediction. A random forest regression (RFR) model for evaluation of nanozyme peroxidase activity is developed and optimized by correlation-based feature selection and hyperparameter tuning, achieving performance up to R2 = 0.796 for Kcat and R2 = 0.627 for Km . Experiment-confirmed unknown nanozyme activity prediction is also demonstrated. Moreover, the DiZyme expandable, open-access resource containing the database, predictive algorithm, and visualization tool is developed to boost novel nanozyme discovery worldwide (https://dizyme.net).

What are inorganic nanozymes? Artificial or inorganic enzymes

Inorganic enzymes, a new class of inorganic nanomaterials with intrinsic enzyme-like properties, are comparable to proteins and RNAs as biocatalysts.

Inorganic Nanozymes: Prospects for Disease Treatments and Detection Applications.

In recent years, with the development of nanomaterials, a slice of nanomaterials has been demonstrated to possess high catalytic activity similar to natural enzymes and counter the dilemmas including easy inactivation and low yield natural of enzymes, which are labeled as nanozymes. The catalytic activity of nanozymes could be easily regulated by size, structure, surface modification and other factors. In comparison with natural enzymes, nanozymes featured with a more stable structure, economical preparation and preservation, diversity of functions and adjustable catalytic activity, thus becoming the potentially ideal substitute for natural enzymes. Generally, the are mainly three types containing metal oxide nanozymes, noble metal nanozymes and carbon-based nanozymes, owing various applications in biomedical, energy and environmental fields. In this review, to summarize the recent representative applications of nanozymes, and potentially explore the scientific problems in this field at the same time, we are going to discuss the catalytic mechanisms of diverse nanozymes, with the emphasis on their applications in the fields of tumor therapy, anti-inflammatory and biosensing, hoping to help and guide the future development of nanozymes.

Bifunctional oxidase-peroxidase inorganic nanozyme catalytic cascade for wastewater remediation

Nanozymes are inorganic nanoparticles with enzyme-like features. Noble metals and metal oxide-based nanomaterials can present enzyme-mimicking behavior mediating catalytic reactions including oxidase-, peroxidase-, catalase-, and superoxide dismutase-like activities with important applications in environmental and biomedical grounds. Thus, in this study, it was designed and developed a novel nanosystem that exploits the oxidase-like (OD) behavior of gold nanoparticles (AuNPs, GOLDzyme) and the peroxidase-like (POD) characteristics of cobalt-doped magnetic iron oxide nanoparticles (MIONs, Co-MIONzyme) amalgamated in an inorganic-inorganic bi-nanozyme cascade for the degradation of dye pollutants. GOLDzyme and Co-MIONzyme nanomaterials were synthesized and stabilized by citrate and carboxymethylcellulose (CMC) ligands, respectively, using a green aqueous colloidal process at mild conditions. The physicochemical characterization results indicated that water-dispersible colloidal supramolecular nanostructures were effectively produced, with core-shell morphologies (i.e., inorganic nanoparticle core/organic-shell). The AuNPs (GOLDzyme) presented crystalline nanostructure, with a uniform spherical shape, zeta potential (ZP) = - 46 ± 3 mV, hydrodynamic diameter (DH) = 11 ± 3 nm. Analogously, the Co-MIONzyme stabilized by CMC ligand evidenced the formation of nanocrystalline substituted magnetite (CoxFe3−xO4) with uniform spherical morphology, average size = 7.0 ± 2 nm, ZP = - 47 ± 3 mV, DH = 46 ± 3 nm, and superparamagnetic behavior. When tested separately using a colorimetric assay based on a chromogenic molecule (3,3′,5,5′ tetramethylbenzidine, TMB), they demonstrated catalytic activity upon the injection of each specific substrate in the medium, i.e., glucose for GOLDzyme that behaved predominantly as oxidase-like nanomaterial, and H2O2 for Co-MIONzyme, which showed a peroxidase-like or catalase-like behavior. In addition, these nanozymes demonstrated pH-dependent and temperature-dependent catalytic activities. As a proof of concept, when combined into a single-pot reaction system, these bifunctional nanozymes confirmed integrated catalytic cascade as oxidase and peroxidase-like nanocatalysts towards the oxidation of TMB and the degradation of methylene blue (MB, ~18%) as the model dye pollutant. Based on the preliminary encouraging results of this research, it can be foreseen that the combination of nanozymes paves the way for the development of new nanoplatforms for prospective applications in water treatment and environmental remediation.

Catalytic ferromagnetic gold nanoparticle immunoassay for the detection and differentiation of Mycobacterium tuberculosis and Mycobacterium bovis

A ferromagnetic gold nanoparticle based immune detection assay, exploiting the enhanced signal amplification of inorganic nanozymes, was developed and evaluated for its potential application in the detection of Mycobacterium tuberculosis complex (MTBC) organisms, and simultaneous identification of Mycobacterium bovis. Ferromagnetic gold nanoparticles (Au–Fe3O4 NPs) were prepared and their intrinsic peroxidase-like activity exploited to catalyse 3,3′,5’,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). When the Au–Fe3O4 NPs were functionalised by direct coupling with MTBC-selective antibodies, a nanoparticle based immune detection assay (NPIDA) was developed which could detect Mycobacterium tuberculosis (MTB) and differentiate M. bovis. In the assay, the intrinsic magnetic capability of the functionalised Au–Fe3O4 NPs was used in sample preparation to capture target bacterial cells. These were incorporated into a novel immunoassay which used species selective monoclonal antibodies (mAb) to detect bound target. The formation of a blue TMB oxidation product, with a peak absorbance of 370 nm, indicated successful capture and identification of the target. The detection limit of the NPIDA for both MTB and M. bovis was determined to be comparable to conventional ELISA using the same antibodies. Although limited matrix effects were observed in either assay, the NPIDA offers a reduced time to confirmatory identification.This novel NPIDA was capable of simultaneous sample concentration, purification, immunological detection and speciation. To our knowledge, it represents the first immune-based diagnostic test capable of identifying MTBC organisms and simultaneously differentiating M. bovis.

L-Cysteine as an Irreversible Inhibitor of the Peroxidase-Mimic Catalytic Activity of 2-Dimensional Ni-Based Nanozymes

The ability to modulate the catalytic activity of inorganic nanozymes is of high interest. In particular, understanding the interactions of inhibitor molecules with nanozymes can bring them one step closer to the natural enzymes and has thus started to attract intense interest. To date, a few reversible inhibitors of the nanozyme activity have been reported. However, there are no reports of irreversible inhibitor molecules that can permanently inhibit the activity of nanozymes. In the current work, we show the ability of L-cysteine to act as an irreversible inhibitor to permanently block the nanozyme activity of 2-dimensional (2D) NiO nanosheets. Determination of the steady state kinetic parameters allowed us to obtain mechanistic insights into the catalytic inhibition process. Further, based on the irreversible catalytic inhibition capability of L-cysteine, we demonstrate a highly specific sensor for the detection of this biologically important molecule.

Targeted self-activating Au-Fe3O4 composite nanocatalyst for enhanced precise hepatocellular carcinoma therapy via dual nanozyme-catalyzed cascade reactions

Catalytic nanozyme-based tumor therapy is currently considered as a promising strategy in anti-tumor treatment, but it also has some disadvantages such as systemic toxicity, off-target, and non-specificity, leading to lowered therapeutic precision and efficacy in clinical curability. To overcome these limitations, herein we report a distinct strategy of nanocatalytic tumor precision therapy via tumor cell-specific CD44 targeted nanozyme-catalyzed cascade reactions. Briefly, the composite nanozyme CD44MMSN/AuNPs were assembled with two self-activable nanocatalysts including the inner core peroxidase-mimic Fe3O4 magnetic nanoparticles (MNPs), and the outer glucose oxidase-mimic AuNPs situated within large aperture mesoporous silicon (MMSN/AuNPs), and then functionalized with cell ligand hyaluronic acid (HA), which can specifically target transmembrane CD44 receptor of HepG2 tumor cells. In terms of catalysis, we found the coupled AuNPs effectively catalyzed glucose to produce H2O2, which was inversely catalyzed by Fe3O4 NPs and AuNPs to produce more hydroxyl radicals (•OH) in tumor microenvironment (TME). That is, this composite nanozyme performed self-activable reactive oxygen species (ROS)-mediated cascade reactions, and thereby resulted in significantly specific growth inhibition of hepatocellular carcinoma HepG2 cells. The present results also showed that the composite nanozyme CD44MMSN/AuNPs could specifically induce a greater number of HepG2 cell apoptosis and death. More importantly, we elucidated the mechanism of the composite nanozyme CD44MMSN/AuNPs via inducing HepG2 cell apoptosis and death from subcellular level. Therefore, this study represents an insightful paradigm for achieving nanocatalytic tumor precision therapy through rationally designing tumor cell-targeting inorganic nanozyme with self-activable cascade reactions.

Intrinsic Peroxidase-Mimicking Ir Nanoplates for Nanozymatic Anticancer and Antibacterial Treatment.

The study of inorganic nanozymes to overcome the disadvantages of bio-enzymes, such as the requirement of optimized reaction conditions and lack of durability against environmental factors, is one of the most significant research topics at present. In this work, we comprehensively analyzed the intrinsic peroxidase-like activity of Ir-based nanoparticles, the biological and nanozymatic potentials of which have not yet been explored. These particles were synthesized by the galvanic replacement of Ag nanoplates with Ir. Through the confirmed peroxidase-like activity and hydrogen peroxide decomposition with free radical generation facilitated by these particles, the antibacterial and anticancer effects were successfully verified in vitro. The nanozyme-based therapeutic effect observed at concentrations at which these nanoparticles do not show cytotoxicity suggests that it is possible to achieve more precise and selective local treatment with these particles. The observed highly efficient peroxidase-like activity of these nanoparticles is attributed to the partially mixed composition of Ir-Ag-IrO2 formed through the galvanic replacement reaction in the synthetic process.

Engineered hybrid nanozyme catalyst cascade based on polysaccharide-enzyme-magnetic iron oxide nanostructures for potential application in cancer therapy

An array of new nanomaterials have been designed to mimic the catalyst characteristics of natural enzymes (termed "nanozymes"), which connects an essential bridge between nanotechnology and biomedical science. Thus, the research on nanozymes has increased dramatically and a new multidisciplinary field has emerged in recent years named nanozymology. Magnetic iron oxide nanoparticles (e.g., Fe2O3 and Fe3O4, MIONs) have revealed to perform an enzyme-like activity with pH-dependent behavior. These nanozymes can catalytically decompose H2O2 into products (e.g., H2O and O2) under mild pH conditions mimicking enzyme-like bioactivity. More importantly, under mildly acidic conditions, they can use H2O2 as the substrate for producing highly toxic reactive oxygen species (ROS) through the generation of hydroxyl radicals (OH), displaying peroxidase-like activity. Therefore, here we designed and developed a novel class of hybrid nanocatalysts based on the conjugation of GOx (natural enzyme) and MIONs (inorganic nanozyme) stabilized by a biocompatible polymer shell of carboxymethyl cellulose. They created supramolecular vesicle-like nanostructures that were tested for killing brain cancer cells (U-87 MG) in vitro, where they proved anticancer activity attributed to ferroptosis-induced cell death. These hybrid nanostructures performed as a cascade of integrated nanocatalysts: a) GOx worked as the starting catalyst, where the glucose in the medium generated H2O2; b) next, H2O2 was catalyzed by the downstream MION nanozymes (Fe3O4) via Fenton-like reactions releasing toxic species (ROS), which caused the cancer cell death. Remarkably, this nanozyme-induced cell death was more pronounced in brain cancer cells ascribed to the more acidic microenvironment than on healthy cells.

Emission Wavelength Switchable Carbon Dots Combined with Biomimetic Inorganic Nanozymes for a Two-Photon Fluorescence Immunoassay

In this work, o-phenylenediamine is utilized as a precursor to synthesize the fluorescent emission wavelength switchable carbon dots (o-CDs). Our investigation reveals that ferrous ions (Fe2+) can effectively induce fluorescence quenching of o-CDs by chelation and aggregation. After the addition of hydrogen peroxide (H2O2), the fluorescence of o-CDs recovers and the fluorescent color changes from yellow to green. As far as we know, o-CDs are the first reported CDs with switchable fluorescence emission wavelength. In order to fabricate an enzyme-free immunosensor, an amino-functionalized dendritic mesoporous silica nanoparticle (DMSN)-gold nanoparticle (Au NP) nanostructure was fabricated as a glucose oxidase mimetic nanoenzyme by in situ coating of the Au NPs on the surface of the DMSNs. Then, the functionalized DMSN-Au NPs were modified on the detection antibody and hydrolyzed with glucose to produce H2O2. This immune induced recognition strategy combines with the o-CDs+Fe2+ signal generation system to achieve specific and sensitive detection of the target. The replacement of glucose oxidase by DMSN-Au NPs not only reduces the cost but also provides significantly amplified signals due to DMSNs haing a high specific surface area. We show the detection of carcinoembryonic antigen (CEA) as an example target to evaluate the analytical figure of merits of the proposed strategy. Under the optimal conditions, two-photon-based o-CDs displayed excellent performances for CEA and the limit of detection as low as 74.5 pg/mL with a linear range from 0.1 to 80 ng/mL. The proposed fluorescent immunosensor provides an optional and potential scheme for low cost, high sensitivity, and versatile discovery of clinical biomarkers.

Novel Imprinted Metallic Nanozymes Based Nanosensor with Fast Catalytic Activity and Specificity for Cortisol

Introduction Cost effective tailoring of inorganic nanoparticles (nanozymes) that mimics enzymatic reaction at diverse physiological condition needs a new approach for sensor development. Developing a feature of enzymes in nanozymes containing analyte selective pockets is challenging. Many reported methods using bio-ligands (aptamers, peptides, antibodies) are defeating the stability and cheap feature of nanozymes [1-3]. To address the challenges in synthesis of enzyme mimicked nanozymes, we proposed multifunctional imprinted metallic nanozymes for selective and catalytic sensing of cortisol. The improved biomimetic detection of cortisol at ppm level in artificial biological samples (blood, saliva) was performed by imprinted metallic nanozymes integrated sensor. Cortisol is the stress biomarker, and provides an option to monitor the physiological and emotional strain [4]. Thus, instant stress can be accurately measured by using the proposed nanosensor. Synthesis of imprinted metallic nanozymes on gold electrode Gold electrode was kept in 20 mL of acid mixture (0.05 mM sulphuric acid and 0.1 mM nitric acid) for 25 minutes. Cleaned gold electrode was used as working electrode, along with Silver electrode (reference electrode) and Platinum wire (counter electrode) for insitu synthesis of imprinted metallic nanozymes using electrochemical method. 50 mL solution containing copper sulphate 0.05 mM) along with aniline (0.05mM) solution at pH 7.4 was electropolymerized for 20 cycles in presence of cortisol (Fig.1 A section (I)). The decrease in the current indicates that the aggregation of nanoparticles increased with the number of scan cycles. Washing of cortisol was performed by ethanol solution to create selective binding pockets in metallic nanozymes. As a control in absence of cortisol, metallic nanozymes synthesis were named for non-imprinted metallic nanozymes (Fig. 1B, section (I)) was also synthesized keeping above mentioned identical experimental condition. Absence of well-defined cortisol peak in cyclic voltammogram (Fig. 1B, section (I)) indicates the formation of non-imprinted metallic nanozymes Morphological Characterization A typical SEM images of imprinted metallic nanozymes with cortisol at lower and higher magnification (Fig. 1A, B, section (II) and metallic nanozymes with binding pockets (Fig. 2C, section (II)) are shown. Gold electrode modified metallic nanoparticles appeared as white droplets with size (70-80 nm) (Fig. 1A section (II)). High magnified SEM image (Fig. 1B, section (II)) reveals the controlled coating of nanozymes on gold electrode. Imprinted metallic nanozyme surface exhibits pores, indicating binding pockets of cortisol. Electrochemical analysis of cortisol in artificial human samples Typical quantitative sensing of cortisol was performed in artificial sweat and blood samples of human in phosphate buffer solution (pH 7.4) using differential pulse voltammetry. The recovery percentage of cortisol on proposed sensor was calculated by standard addition method [5]. The obtained recovery results for both samples are tabulated in Table 1. Cortisol recovery % revealed that the proposed sensor could be used as wearable device for cortisol analysis in sweat. Conclusions Inorganic nanozymes could provide cost effective, scalable sensing platform with good selectivity towards cortisol. Selective binding pockets for cortisol were successfully developed in nanozymes with good catalytic property and recovery. Obtained results insights into easy monitoring of cortisol and enables the new approach of developing scalable sensor with improve accuracy. These excellent properties of proposed nanosensor could be useful for fabrication of a light weight flexible reusable wearable device.