Nanoisozymes: Crystal‐Facet‐Dependent Enzyme‐Mimetic Activity of V2O5 Nanomaterials

Nanomaterials with enzyme‐like activity (nanozymes) attract significant interest owing to their applications in biomedical research. Particularly, redox nanozymes that exhibit glutathione peroxidase (GPx)‐like activity play important roles in cellular signaling by controlling the hydrogen peroxide (H₂O₂) level. Herein we report, for the first time, that the redox properties and GPx‐like activity of V₂O₅ nanozyme depends not only on the size and morphology, but also on the crystal facets exposed on the surface within the same crystal system of the nanomaterials. These results suggest that the surface of the nanomaterials can be engineered to fine‐tune their redox properties to act as “nanoisozymes” for specific biological applications.     

3,4,9,10‐Perylenetetracarboxylic Acid/Hemin Nanocomposites Act as Redox Probes and Electrocatalysts for Constructing a Pseudobienzyme‐Channeling Amplified Electrochemical Aptasensor

A simple wet‐chemical strategy for the synthesis of 3,4,9,10‐perylenetetracarboxylic acid (PTCA)/hemin nanocomposites through π–π interactions is demonstrated. Significantly, the hemin successfully conciliates PTCA redox activity with a pair of well‐defined redox peaks and intrinsic peroxidase‐like activity, which provides potential application of the PTCA self‐derived redox activity as redox probes. Additionally, PTCA/hemin nanocomposites exhibit a good membrane‐forming property, which not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer. On the basis of these unique properties, a pseudobienzyme‐channeling amplified electrochemical aptasensor is developed that is coupled with glucose oxidase (GOx) for thrombin detection by using PTCA/hemin nanocomposites as redox probes and electrocatalysts. With the addition of glucose to the electrolytic cell, the GOx on the aptasensor surface bioelectrocatalyzed the reduction of glucose to produce H₂O₂, which in turn was electrocatalyzed by the PTCA/hemin nanocomposites. Cascade schemes, in which an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor sensitivity. As a result, a linear relationship for thrombin from 0.005 to 20 nM and a detection limit of 0.001 nM were obtained.     

Filling Carbon Nanotubes with Prussian Blue Nanoparticles of High Peroxidase‐Like Catalytic Activity for Colorimetric Chemo‐ and Biosensing

Facile filling of multiwalled carbon nanotubes (MWCNTs) with Prussian blue nanoparticles (PBNPs) of high peroxidase‐like catalytic activity was performed to develop novel colorimetric sensing protocols for assaying H₂O₂ and glucose. Fine control of PBNP growth was achieved by modulating the concentration ratio of K₃[Fe(CN)₆] and FeSO₄ precursors in an acidic solution containing ultrasonically dispersed MWCNTs, and thus size‐matched PBNPs could be robustly immobilized in the cavities of the MWCNTs (MWCNT‐PB_in). Unlike other reported methods involving complicated procedures and rigorous preparation/separation conditions, this mild one‐pot filling method has advantages of easy isolation of final products by centrifugation, good retention of the pristine outer surface of the MWCNT shell, and satisfactory filling yield of (24±2) %. In particular, encapsulation of PBNPs of poor dispersibility and limited functionality in dispersible and multifunctional MWCNT shells creates new and valuable opportunities for quasihomogeneous‐phase applications of PB in liquid solutions. The MWCNT‐PB_in nanocomposites were exploited as a peroxidase mimic for the colorimetric assay of H₂O₂ in solution by using 3,3′,5,5′‐tetramethylbenzidine (TMB) as reporter, and they gave a linear absorbance response from 1 μM to 1.5 mM with a limit of detection (LOD) of 100 nM . Moreover, glucose oxidase (GOx) was anchored on the outer surface of MWCNT‐PB_in to form GOx/MWCNT‐PB_in bionanocomposites. The cooperation of outer‐surface biocatalysis with peroxidase‐like catalysis of interior PB resulted in a novel cooperative colorimetric biosensing mode for glucose assay. The use of GOx/MWCNT‐PB_in for colorimetric biosensing of glucose gave a linear absorbance response from 1 μM to 1.0 mM and an LOD of 200 nM . The presented protocols may be extended to other multifunctional nanocomposite systems for broad applications in catalysis and biotechnology.     

FeS2−AuNPs Nanocomposite as Mimicking Enzyme for Constructing Signal‐off Sandwich‐type Electrochemical Immunosensor Based on Electroactive Nickel Hexacyanoferrate as Matrix

In this work, a novel sandwich‐type electrochemical immunosensor with electroactive nickel hexacyanoferrate nanoparticles (NiHCFNPs) as matrix was constructed for α‐fetoprotein (AFP) detection in a signal‐off manner by using FeS₂?AuNPs nanocomposite catalyzed insoluble precipitation to significantly inhibit the electrochemical signal. Initially, the NiHCFNPs with excellent electrochemical property was modified on the electrodeposited nano‐Au electrode to obtain a strong initial electrochemical signal. Subsequently, another nano‐Au layer was formed for immobilization of capture antibody (Ab₁). In the presence of target AFP, the prepared FeS₂?AuNPs‐Ab₂ bioconjugate could be specifically recognized and immobilized on electrode through the sandwich‐type immunoreaction. The FeS₂ with large specific surface areas were used as scaffolds to load abundant mimicking enzyme AuNPs. With the help of hydrogen peroxide (H₂O₂), FeS₂?AuNPs with peroxidase‐like activity accelerated the 4‐chloro‐1‐naphthol (4‐CN) oxidation with generation of insoluble precipitation on electrode, which would greatly hinder the electron transfer and thus caused the decrease of electrochemical signal for quantitative determination of AFP. This approach achieved a wide dynamic linear range from 0.0001 to 100 ng mL^?1 with an ultralow limit detection of 0.028 pg mL^?1. Especially, the proposed AFP immunosensor can be applied to detect human serum samples with satisfactory results, indicating a potential application in clinical monitoring of tumor biomarkers.     

A Redox Modulatory Mn3O4 Nanozyme with Multi‐Enzyme Activity Provides Efficient Cytoprotection to Human Cells in a Parkinson's Disease Model

Nanomaterials with enzyme‐like activities (nanozymes) attracts significant interest due to their therapeutic potential for the treatment of various diseases. Herein, we report that a Mn₃O₄ nanozyme functionally mimics three major antioxidant enzymes, that is, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the multienzyme activity is size as well as morphology‐dependent. The redox modulatory effect of Mn₃O₄ plays a crucial role in protecting the cells from MPP+ induced cytotoxicity in a Parkinson disease (PD)‐like cellular model, indicating that manganese‐based nanomaterials having multi‐enzyme activity can robustly rescue the cells from oxidative damage and thereby possess therapeutic potential to prevent ROS‐mediated neurological disorders.     

Control of Biocatalytic Transformations by Programmed DNA Assemblies

This study demonstrates the self‐assembly of inhibitor/enzyme‐tethered nucleic acid fragments or enzyme I‐, enzyme II‐modified nucleic acids into functional nanostructures that lead to the controlled inhibition of the enzyme or the activation of an enzyme cascade. In one system, the anti‐cocaine aptamer subunits are modified with monocarboxy methylene blue (MB⁺) as the inhibitor and with choline oxidase (ChOx). The cocaine‐induced self‐assembly of the aptamer subunits complex results in the inhibition of ChOx by MB⁺. In a further configuration, two nucleic acids of limited complementarity are functionalized at their 3′ and 5′ ends with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, or with MB⁺ and ChOx. In the presence of a target DNA sequence, synergistic complementary base‐pairing occurs, thus leading to stable supramolecular Y‐shaped nanostructures of the nucleic acid units. A GOx/HRP bienzyme cascade or the programmed inhibition of ChOx by MB⁺ is demonstrated in the resulting nucleic acid nanostructures. A quantitative theoretical model that describes the nucleic acid assemblies and that results in the inhibition of ChOx by MB⁺ or in the activation of the GOx/HRP cascade, respectively, is provided.     

Glucose‐Responsive Sequential Generation of Hydrogen Peroxide and Nitric Oxide for Synergistic Cancer Starving‐Like/Gas Therapy

Glucose is a key energy supplier and nutrient for tumor growth. Herein, inspired by the glucose oxidase (GOx)‐assisted conversion of glucose into gluconic acid and toxic H₂O₂, a novel treatment paradigm of starving‐like therapy is developed for significant tumor‐killing effects, more effective than conventional starving therapy by only cutting off the energy supply. Furthermore, the generated acidic H₂O₂ can oxidize l ‐Arginine (l ‐Arg) into NO for enhanced gas therapy. By using hollow mesoporous organosilica nanoparticle (HMON) as a biocompatible/biodegradable nanocarrier for the co‐delivery of GOx and l ‐Arg, a novel glucose‐responsive nanomedicine (l ‐Arg‐HMON‐GOx) has been for the first time constructed for synergistic cancer starving‐like/gas therapy without the need of external excitation, which yields a remarkable H₂O₂–NO cooperative anticancer effect with minimal adverse effect.     

Switchable Enzyme/DNAzyme Cascades by the Reconfiguration of DNA Nanostructures

Mimicking cellular transformations and signal transduction pathways by means of biocatalytic cascades proceeding in organized media is a scientific challenge. We describe two DNA machines that enable the “ON/OFF” switchable activation and deactivation of three‐component biocatalytic cascades. One system consists of a reconfigurable DNA tweezers‐type structure, whereas in the second system the catalytic cascade proceeds on a switchable DNA clamp scaffold. The three‐component catalytic cascades consist of β‐galactosidase (β‐Gal), glucose oxidase (GOx), and the K⁺‐ion‐stabilized hemin‐G‐quadruplex horseradish peroxidase (HRP)‐mimicking DNAzyme. The hemin‐G‐quadruplex‐bridged closed structure of the tweezers or clamp allows the biocatalytic cascades to operate (switched “ON′′), whereas separation of the hemin‐G‐quadruplex by means of 18‐crown‐6‐ether opens the tweezers/clamp structures, thus blocking the catalytic cascade (switched ”OFF“). This study is complemented by two‐component, switchable biocatalytic cascades composed of GOx and hemin‐G‐quadruplex assembled on hairpin‐bridged DNA tweezers or clamp nanostructures.     

纳米材料修饰电极上吸附态葡萄糖氧化酶的酶活性和电活性的比较

采用石英晶体微天平(QCM)技术, 监测了裸金(Au)电极、电沉积纳米金的金电极(Au_ed/Au)、多壁碳纳米管(MWCNTs)修饰的金电极(MWCNTs/Au)以及MWCNTs 修饰后再电沉积纳米金的金电极(Au_ed/MWCNTs/Au)上葡萄糖氧化酶(GOx)的吸附过程, 测算了吸附固定的GOx质量. 通过阳极恒电位检测吸附酶与葡萄糖发生酶反应所产生的过氧化氢, 考察了这些酶电极的安培响应, 并测算了各吸附态GOx的质量比生物活性(MSBAi).也通过循环伏安法研究酶的直接电化学, 测算了各吸附态GOx的电活性百分数(EAP_i). 实验结果表明, 酶吸附量和酶电极的安培响应满足MWCNTs/Au > Au_ed/MWCNTs/Au > Au_ed/Au > Au 的顺序; MSBA_i满足Au > Au_ed/MWCNTs/Au > Au_ed/Au > MWCNTs/Au的顺序; 而EAP_i则满足MWCNTs/Au > Au_ed/MWCNTs/Au > Au_ed /Au > Au的顺序. 根据酶和纳米材料的亲疏水作用以及酶的吸附量对实验结果进行了合理解释, 也定量验证了电极上吸附酶分子的总生物活性与酶电极的安培响应呈正相关关系, 所得数据和结论有助于纳米材料固定酶及其安培酶电极的研究.     

Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on‐column enzymatic assay

A highly specific and sensitive method for glucose quantification in human serum samples based on on‐column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter‐microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2‐[6‐(4′‐amino) phenoxy‐3H‐xanthen‐3‐on‐9‐yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H₂O₂) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α‐glucosidase activity and screening for its inhibitors.     

Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H₂O₂ and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM^?1 cm^?2) and stability (80% remains after 30 days).     

3,4‐Dihydroxy‐L‐phenylalanine for Preparation of Gold Nanoparticles and as Electron Transfer Promoter in H2O2 Biosensor

3,4‐Dihydroxy‐L ‐phenylalanine (dopa) and 2‐(3,4‐dihydroxyphenyl)ethylamine (dopamine) were investigated as reducing agent and stabilizer for synthesis of gold nanoparticles (AuNPs) by one‐pot heating of a solution of HAuCl₄/dopa or dopamine. AuNPs with different sizes were obtained by controlling the mass ratios of HAuCl₄/dopa or dopamine. The formation mechanism for AuNPs was also proposed. Immobilization of horseradish peroxidase (HRP) and promotion of its electron transfer by polydopa film were investigated for preparation of H₂O₂ biosensor. Alkaline dopa solution was dropped onto a gold electrode for the formation of polydopa film. HRP was immobilized on the polydopa film through interactions between heme centre of HRP and the amine and carboxyl groups in polydopa. The AuNPs embedded in the polydopa film improved the electron transfer efficiency. These two factors allowed successful development of a H₂O₂ sensor with HRP@polydopa‐AuNPs electrode. Due to its biocompatibility, the polydopa‐AuNPs film provided good retention of enzyme activity and long‐term stability of the sensor. A rapid catalytic response (3 s) and a linear range from 0.006 to 5.0 mmol L^?1 were obtained for H₂O₂. This facile preparation strategy can be extended to other enzyme‐based biosensors.     

Synthesis and catalytic evaluation of Fe3O4/MWCNTs nanozyme as recyclable peroxidase mimetics: Biochemical and physicochemical characterization

In this research, the nanocomposite of multiwalled carbon nanotubes and magnetic metal oxide nanoparticles (Fe₃O₄/MWCNTs), as enzyme mimetic, was synthesized using an in situ chemical reduction method. The structure, composition and morphology of the prepared Fe₃O₄/MWCNT nanocomposite materials were characterized using X‐ray diffraction, FT‐IR and scanning electron microscopy with energy dispersive X‐ray spectroscopy, respectively. The magnetic properties of the nanocomposite were investigated by the vibrating sample magnetometer. A colorimetric system involving nanozyme, phenol/4‐aminoantipyrine and H₂O₂ was utilized for the determination of peroxidase mimetic catalytic assay. The obtained results confirmed that the synthesis of Fe₃O₄/MWCNTs nanostructures was successful. It was found that Fe₃O₄/MWCNTs nanohybrid exhibited peroxidase‐like activity without any pH limitation. Colorimetric data demonstrated that the prepared nanocatalyst had higher catalytic activity toward H₂O₂ than MWCNTs. The kinetic parameters of the nanozyme, K_m and V_max, were estimated to be 8.3 mm and 1.4 mm min^?1, respectively. The Fe₃O₄/MWCNTs nanostructures were also successfully applied for glucose detection. In addition, peroxidase‐like activity of the nanozyme increased in the presence of butyl‐imidazolium bromide ionic liquid. These biomimetic catalysts have some advantages, such as simplicity, stability, reusability and cost effectiveness, which makes them great candidates to be used in various fields of biotechnology applications.     

Liposomal Enzyme Nanoreactors Based on Nanoconfinement for Efficient Antitumor Therapy

Enzymatic reactions can consume endogenous nutrients of tumors and produce cytotoxic species and are therefore promising tools for treating malignant tumors. Inspired by nature where enzymes are compartmentalized in membranes to achieve high reaction efficiency and separate biological processes with the environment, we develop liposomal nanoreactors that can perform enzymatic cascade reactions in the aqueous nanoconfinement of liposomes. The nanoreactors effectively inhibited tumor growth in vivo by consuming tumor nutrients (glucose and oxygen) and producing highly cytotoxic hydroxyl radicals (⋅OH). Co-compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP) in liposomes could increase local concentration of the intermediate product hydrogen peroxide (H₂O₂) as well as the acidity due to the generation of gluconic acid by GOx. Both H₂O₂ and acidity accelerate the second-step reaction by HRP, hence improving the overall efficiency of the cascade reaction. The biomimetic compartmentalization of enzymatic tandem reactions in biocompatible liposomes provides a promising direction for developing catalytic nanomedicines in antitumor therapy.     

GOx@ZIF‐8(NiPd) Nanoflower: An Artificial Enzyme System for Tandem Catalysis

We report a facile approach to prepare an artificial enzyme system for tandem catalysis. NiPd hollow nanoparticles and glucose oxidase (GOx) were simultaneously immobilized on the zeolitic imidazolate framework 8 (ZIF‐8) via a co‐precipitation method. The as‐prepared GOx@ZIF‐8(NiPd) nanoflower not only exhibited the peroxidase‐like activity of NiPd hollow nanoparticles but also maintained the enzymatic activity of GOx. A colorimetric sensor for rapid detection of glucose was realized through the GOx@ZIF‐8(NiPd) based multi‐enzyme system. Moreover, the GOx@ZIF‐8(NiPd) modified electrode showed good bioactivity of GOx and high electrocatalytic activity for the oxygen reduction reaction (ORR), which could also be used for electrochemical detection of glucose.     

Guanine/Ionic Liquid Derived Ordered Mesoporous Carbon Decorated with AuNPs as Efficient NADH Biosensor and Suitable Platform for Enzymes Immobilization and Biofuel Cell Design

Guanine‐ionic liquid derived ordered mesoporous carbon (GIOMC) decorated with gold nanoparticles was used as electrocatalyste for NADH oxidation and electrochemical platform for immobilization of glucose dehydrogenase (GDH) enzyme. The resulting GIOMC/AuNPs on the glassy carbon electrode can be used as novel redox‐mediator free for NADH sensing and this integrated system (GIOMC/AuNPs/GDH) shows excellent electrocatalytic activity toward glucose oxidation. Furthermore, the ionic liquid derived ordered mesoporous carbon derivate with Ph‐SO₃H (IOMC‐PhSO₃H) decorated with AuNPs has been developed to bilirubin oxidase enzyme (BOD) immobilization and the GC/IOMC‐PhSO₃H/BOD integrated system shows excellent bioelectrocatalytic activity toward oxygen reduction reaction. The proposed mesostructured platforms decorated by AuNPs have been developed to enhance mass transfer and charge transfer from biocatalyst to electrode, leading these bioanode and biocathode used for biofuel cell assembly. Integration designed bioanode and biocathode yielding a membrane‐less glucose/O₂ biofuel cell with power density of 33 (mW.cm⁻²) at 257 mV. The open circuit voltage of this biofuel cell and maximum produced current density were 508 mV and 0.252 (mA.cm⁻²) respectively.     

Self‐Assembly of Multi‐nanozymes to Mimic an Intracellular Antioxidant Defense System

In this work, for the first time, we constructed a novel multi‐nanozymes cooperative platform to mimic intracellular antioxidant enzyme‐based defense system. V₂O₅ nanowire served as a glutathione peroxidase (GPx) mimic while MnO₂ nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V₂O₅@pDA@MnO₂ nanocomposite could serve as one multi‐nanozyme model to mimic intracellular antioxidant enzyme‐based defense procedure in which, for example SOD, CAT, and GPx co‐participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy.     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.     

Electrochemical Behaviors of Glucose Oxidase Based on Biocatalytic Deposition of Gold Nanoparticles

Coupling nanotechnology with biocatalysis, a highly sensitive glucose biosensor for the study of electrochemical behaviors of glucose oxidase (GOx) was proposed by using monkshoodvine root–bark like carbon (MLC) as the platform for the biocatalytic deposition of AuNPs. The biosensor showed a linear range from 0.25 to 130 μM with a detection limit of 0.1 μM (S/N = 3) towards glucose and sensitivity of 3010 μA/mM. K value was calculated to be 67.4 μM. Furthermore, the proposed AuNPs/GOx–MLC modified pyrolytic graphite electrode (AuNPs/GOx–MLC/PGE) achieved direct electron transfer of GOx. Γ* was calculated to be 2.79 × 10^?11 mol/cm² and k_s was 1.79 s^?1. It also showed a remarkable electrocatalysis towards glucose.     

Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole

Graphite rod (GR) modified with electrochemicaly deposited gold nanoparticles (AuNPs) and adsorbed glucose oxidase (GOx) was used in amperometric glucose biosensor design. Enzymatic formation of polypyrrole (Ppy) on the surface of GOx/AuNPs/GR electrode was applied in order to improve analytical characteristics and stability of developed biosensor. The linear glucose detection range for Ppy/GOx/AuNPs/GR electrode was dependent on the duration of Ppy‐layer formation and the linear interval was extended up to 19.9 mmol L⁻¹ after 21 h lasting synthesis of Ppy. The sensitivity of the developed biosensor was determined as 21.7 μA mM⁻¹ cm⁻², the limit of detection – 0.20 mmol L⁻¹. Ppy/GOx/AuNPs/GR electrodes demonstrated advanced good stability (the t _1/2 was 9.8 days), quick detection of glucose (within 5 s) in the wide linear interval. Additionally, formed Ppy layer decreased the influence of electroactive species on the analytical signal. Developed biosensor is suitable for the determination of glucose in human serum samples.