Organic-inorganic Hybrid Nanoflowers Research Articles

Evaluation of the Antimicrobial Activity of Triple Enzyme-Embedded Organic-Inorganic Hybrid Nanoflowers (hNFs) in Comparison with Powerful Antimicrobial Agent Chitosan.

Organic-inorganic hybrid nanoflowers (hNFs) have high stability, reusability, low production cost, and overcome substrate/product inhibition. Antimicrobial activity of various hNFs has been reported to overcome environmental microbial contaminations and infections, which are considered major public health problems. α-amylase, protease, and lipase are the most common industrial enzymes exerting antimicrobial activity; therefore, we synthesized triple enzyme (α-amylase, protease, and lipase)-embedded hNFs by using pancreatin to evaluate their antimicrobial activity in comparison with one of the most potent antimicrobial polymer chitosan. The broad spectrum of the antimicrobial properties of chitosan is used in industrial products, including cosmetics, food, agriculture, pharmaceuticals, and textiles. SEM analysis, thermogravimetric analysis (TGA), and the degree of deacetylation (%DD) were performed for chitosan characterization, where SEM, FTIR, EDX, and XRD analyses were performed for the characterization of hNFs. The catalytic activity of pancreatin and hNFs was evaluated by measuring lipase, α-amylase, and protease enzyme activities at 37°C. Antibacterial activities of hNFs, pancreatin, and chitosan were tested on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria, compared to the pancreatin and chitosan via agar and broth dilution methods. hNFs showed enhanced catalytic activity for protease, lipase, and α-amylase compared to pancreatin at different pH values (pH 8, 9). hNFs showed catalytic activity after being washed and reused up to six times, indicating their reusability and recoverability. hNFs showed significant antimicrobial activity, such as chitosan, Staphylococcus aureus, and Escherichia coli, compared to pancreatin. Our novel hNFs can be used to develop antimicrobial technologies to fight against environmental microbial contaminations and antibiotic resistance-driven environmental pathogens.

Biosynthesis of Naringenin@Cu (II), Zn (II) Hybrid Nanoflower: Anticancer Activity Assay process

In the study, the synthesis of organic-inorganic hybrid nanoflowers, which have a flower-like structure and are called nanoflowers, was carried out. For this purpose, hybrid nanoflower synthesis was carried out using naringenin as the organic part and different metal ions (Cu and Zn) as the inorganic part. Various analyzes were carried out to characterize the synthesized nanoflowers (such as SEM, EDX, FTIR, XRD). In addition, the anticancer activities of hybrid nanoflowers were tested on the MCF7 (breast cancer) cell line. It was concluded that there was a good increase in anticancer activity when naringenin was converted into hybrid nanoflower form. In particular, the anticancer activity of hybrid nanoflowers synthesized with Zn metal ion was quite excellent. This means that Zn hybrid nanoflowers may be a safer therapeutic alternative than others.

In situ growth of double-hybrid nanoflowers on paper for the visual detection of carcinoembryonic antigen

In this study, the hemoglobin, egg white, copper phosphate organic–inorganic hybrid nanoflowers (H-E-Cu-NFs), and filter paper-based-egg white-Ca3(PO4)2 HNFs (FP-E-Ca-NFs) were synthesized using a mild biomineralization method. Antibodies (Ab) were labeled on the surface of nanoflowers through highly specific interactions between avidin and biotin. The FP-E-Ca-NFs@Biotin N-hydroxysuccinimide ester–Anti-carcinoembryonic antigen primary antibodies (Biotin-NHS-Ab1)/carcinoembryonic antigen (CEA)/H-E-Cu-NFs@Biotin-NHS-Ab2 immune-biosensing system was constructed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to achieve the visual detection of CEA. Among them, H-E-Cu-NFs have abundant enzyme-like catalytic sites of Fe3+/Cu2+, which can effectively improve the efficiency of the colorimetric reaction. In contrast, E-Ca-NFs have no catalytic sites, and the directional design of catalytic performance realizes the specific identification and signal amplification of CEA detection. The as-developed immune-biosensing system showed a detection range of 0.5 ∼ 100 ng/mL, with the lowest detection limit of 0.5 ng/mL and the CEA recovery between 89.5 % ∼ 108.1 % in human serum samples. Compared to traditional ELISA methods, this immune-biosensing system has the advantages of easy operation, low cost, rapid detection, and on-site detection.

Nanoflower Microreactor Based Versatile Enhancer for Recognition Cofactor-Dependent Enzyme Biocatalysis toward Saxitoxin Detection.

Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.

Immobilization of Arginase Using the Organic-Inorganic Hybrid Nanoflower Strategy for L-Ornithine Production

Immobilization of Arginase Using the Organic-Inorganic Hybrid Nanoflower Strategy for L-Ornithine Production

The Formation of D-Allulose 3-Epimerase Hybrid Nanoflowers and Co-Immobilization on Resins for Improved Enzyme Activity, Stability, and Processability.

As a low-calorie sugar, D-allulose is produced from D-fructose catalyzed by D-allulose 3-epimerase (DAE). Here, to improve the catalytic activity, stability, and processability of DAE, we reported a novel method by forming organic-inorganic hybrid nanoflowers (NF-DAEs) and co-immobilizing them on resins to form composites (Re-NF-DAEs). NF-DAEs were prepared by combining DAE with metal ions (Co2+, Cu2+, Zn2+, Ca2+, Ni2+, Fe2+, and Fe3+) in PBS buffer, and were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. All of the NF-DAEs showed higher catalytic activities than free DAE, and the NF-DAE with Ni2+ (NF-DAE-Ni) reached the highest relative activity of 218%. The NF-DAEs improved the thermal stability of DAE, and the longest half-life reached 228 min for NF-DAE-Co compared with 105 min for the free DAE at 55 °C. To further improve the recycling performance of the NF-DAEs in practical applications, we combined resins and NF-DAEs to form Re-NF-DAEs. Resins and NF-DAEs co-effected the performance of the composites, and ReA (LXTE-606 neutral hydrophobic epoxy-based polypropylene macroreticular resins)-based composites (ReA-NF-DAEs) exhibited outstanding relative activities, thermal stabilities, storage stabilities, and processabilities. The ReA-NF-DAEs were able to be reused to catalyze the conversion from D-fructose to D-allulose, and kept more than 60% of their activities after eight cycles.

Enzymes encapsulated in organic–inorganic hybrid nanoflower with spatial localization for sensitive and colorimetric detection of formate

Formate is an important environmental pollutant, and meanwhile its concentration change is associated with a variety of diseases. Thus, rapid and sensitive detection of formate is critical for the biochemical analysis of complex samples and clinical diagnosis of multiple diseases. Herein, a colorimetric biosensor was constructed based on the cascade catalysis of formate oxidase (FOx) and horseradish peroxidase (HRP). These two enzymes were co-immobilized in Cu3(PO4)2-based hybrid nanoflower with spatial localization, in which FOx and HRP were located in the shell and core of nanoflower, respectively (FOx@HRP). In this system, FOx could catalyze the oxidation of formate to generate H2O2, which was then utilized by HRP to oxidize 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid to yield blue product. Ideal linear correlation could be obtained between the absorbance at 420 nm and formate concentration. Meanwhile, FOx@HRP exhibited excellent detection performance with low limit of detection (6 μM), wide linear detection range (10–900 μM), and favorable specificity, stability and reusability. Moreover, it could be applied in the detection of formate in environmental, food and biological samples with high accuracy. Collectively, FOx@HRP provides a useful strategy for the simple and sensitive detection of formate and is potentially to be used in biochemical analysis and clinical diagnosis.

Recent Advances in the Strategies of Simultaneous Enzyme Immobilization Accompanied by Nanocarrier Synthesis

In recent years, with advancements in nanotechnology and materials science, new enzyme immobilization strategies based on nanomaterials have continuously emerged. These strategies have shown significant effects on enhancing enzyme catalytic performance and stability due to their high surface area, good chemical stability, and ease of enzyme binding, demonstrating tremendous potential for industrial applications. Those methods that can rapidly synthesize nanocarriers under mild conditions allow for the one-step synthesis of nanocarriers and enzyme complexes, thereby exhibiting advantages such as simplicity of process, minimal enzyme damage, short processing times, and environmental friendliness. This paper provides an overview of simultaneous enzyme immobilization strategies accompanied by nanocarrier synthesis, including organic–inorganic hybrid nano-flowers (HNFs), metal–organic frameworks (MOFs), and conductive polymers (CPs). It covers their preparation principles, post-immobilization performance, applications, and existing challenges.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone

In this study, we prepared a functional organic–inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme’s terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 ∼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3–11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

Development of enzyme-inorganic hybrid nanoflower-modified electrodes and a smartphone-controlled electrochemical analyzer for point-of-care testing of salivary amylase in saliva.

Quantitation of salivary alpha-amylase (sAA) plays a significant role in not only theoretical studies but also clinical practice. This study reports a quantitative point-of-care testing (POCT) system for sAA quantitation anywhere, anytime and by anyone, which consists of customized electrodes and a smartphone-controlled electrochemical analyzer. Organic-inorganic hybrid nanoflowers (NFs) encapsulating α-glucosidase (AG) and glucose dehydrogenase (GDH) have been synthesized and modified onto screen-printed electrodes (SPCEs) to fabricate the customized electrodes. The SPCEs integrated with the smartphone-controlled electrochemical analyzer exhibit good analytical performance for sAA with a low detection limit of 5.02 U mL-1 and a wide dynamic range of 100-2000 U mL-1 using chronoamperometry. The reported POCT system has been successfully demonstrated for quantitation of sAA in clinical saliva samples, and the quantitation results correlated well with those of the Bernfeld method which is extensively used in clinics. More importantly, this study reveals the great potential of sAA as an early warning indicator of abnormal glucose metabolism in obese individuals. Considering the non-invasive saliva sampling process as well as the easy-to-use and cost-effectiveness features of this quantitative POCT system, quantitation of salivary sAA at home by laypersons might become an appealing choice for obese individuals to monitor their glucose metabolism status anytime.

Organic–inorganic hybrid nanoflowers as a new biomimetic platform for ROS-induced apoptosis by photodynamic therapy

We report here a newly and facile synthesis of the phospholipids@gold nanoflowers (AuNFs) from intact cells as a new biomimetic organic-inorganic hybrid. The most appealing feature of this nanostructure is its dual-absorbing peak in near infrared (NIR) and visible region of spectra, which makes them a potential light-sensitive agent for reactive oxygen species (ROS)-induced apoptosis. Here, in contrast to previous studies, proposed nanostructures are synthesized in a one-pot reaction using phospholipids present in living cell membranes (as a donor cell) with detectable micro process of AuNF formation. The properties of the resulting AuNFs were evaluated through transmission electron microscopy (TEM), as well as FT-IR, 31P-NMR spectra and UV–Vis spectroscopy. Designed cell membrane-based nanostructure looks like an intact cell and would be able to interact with other cells (as a target cell) and also capable to produce cytotoxic singlet oxygen under NIR irradiation. Generated ROS act as a key player in initiation of programmed cell death (apoptosis) and progress of cancer photodynamic therapy (PDT). Cellular experiments on breast cancer MCF-7 cells demonstrated that they may be effective as photodynamic therapy agents.

Controllable Synthesis of Hemoglobin-Metal Phosphate Organic-Inorganic Hybrid Nanoflowers and Their Applications in Biocatalysis.

In recent years, organic-inorganic hybrid nanoflower technology has become an effective method for enzyme immobilization. Here, seven hierarchical flower-like hemoglobin-phosphate organic-inorganic hybrid nanomaterials (Hb-M3(PO4)2·nH2O HNFs) were synthesized through an improved universal one-pot wet-chemical method, with Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ as inorganic components. In this synthesis process, the metal cations are successively involved in the coordination reaction with Hb and the metathesis reaction to generate phosphate precipitation. The coordination ability of metal cations and the generation rate of phosphate precipitations were evaluated, then the progress of the two chemical reactions was controlled synchronously by adjusting the phosphate buffer (PB) concentration, and finally a flower-like structure conducive to substrate diffusion and transport was obtained. Due to the conformational transformation of hemoglobin and the abundant Cu2+/Fe3+ active sites, the hemoglobin-Cu3(PO4)2·3H2O nanoflowers have extremely high catalytic activity, which is ∼14 times that of Hb. Importantly, this method is suitable for the monometallic-ionic, polymetallic-ionic and polyvalent metal-ion nanoflowers, which broadens the chemical composition and structural diversity of nanoflowers.

Preparation of Alcohol Dehydrogenase-Zinc Phosphate Hybrid Nanoflowers through Biomimetic Mineralization and Its Application in the Inhibitor Screening.

A biomimetic mineralization method was used in the facile and rapid preparation of nanoflowers for immobilizing alcohol dehydrogenase (ADH). The method mainly uses ADH as an organic component and zinc phosphate as an inorganic component to prepare flower-like ADH/Zn3(PO4)2 organic-inorganic hybrid nanoflowers (HNFs) with the high specific surface area through a self-assembly process. The synthesis conditions of the ADH HNFs were optimized and its morphology was characterized. Under the optimum enzymatic reaction conditions, the Michaelis-Menten constant (Km) of ADH HNFs (β-NAD+ as substrate) was measured to be 3.54 mM, and the half-maximal inhibitory concentration (IC50) of the positive control ranitidine (0.2-0.8 mM) was determined to be 0.49 mM. Subsequently, the inhibitory activity of natural medicine Penthorum chinense Pursh and nine small-molecule compounds on ADH was evaluated using ADH HNFs. The inhibition percentage of the aqueous extract of P. chinense is 57.9%. The vanillic acid, protocatechuic acid, gallic acid, and naringenin have obvious inhibitory effects on ADH, and their percentages of inhibition are 55.1%, 68.3%, 61.9%, and 75.5%, respectively. Moreover, molecular docking analysis was applied to explore the binding modes and sites of the four most active small-molecule compounds to ADH. The results of this study can broaden the application of immobilized enzymes through biomimetic mineralization, and provide a reference for the discovery of ADH inhibitors from natural products.

Preparation and investigation of novel endopeptidase-exopeptidase co-immobilized nanoflowers with improved cascade hydrolysis

Enzymatic hydrolysis is a promising approach for protein and food processing. However, the efficiency of this approach is constrained by the self-hydrolysis, self-agglomeration of free enzymes and the limited applicability resulted from enzymes' selectivityt. Here, novel organic-inorganic hybrid nanoflowers (AY-10@AXH-HNFs) were prepared by coordinating Cu2+ with both endopeptidase of PROTIN SD-AY10 and exopeptidase of Prote AXH. The results indicate that the AY-10@AXH-HNFs exhibited 4.1 and 9.6 times higher catalytic activity than free Prote AXH and PROTIN SD-AY10, respectively, for the enzymatic hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE). The kinetic parameters of Km, Vmax and Kcat/Km by AY-10@AXH-HNFs were determined to be 0.6 mg/mL, 6.8 mL·min/mg and 6.1 mL/(min·mg), respectively, surpassing the values obtained from free endopeptidase and exopeptidase. Furthermore, the ability of AY-10@AXH-HNFs to retain 41 % of their initial catalytic activity after undergoing 5 cycles of repeated use confirmed their stability and reusability. This study introduces a novel approach of co-immobilizing endopeptidase and exopeptidase on nanoflowers, resulting in significantly enhanced stability and reusability of the protease in catalytic applications.

Introduction, synthesis procedures, and applications of organic-inorganic hybrid nanoflowers in biosciences

Introduction, synthesis procedures, and applications of organic-inorganic hybrid nanoflowers in biosciences

Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was defined as 4.3 CFU mL−1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.

In Situ Synthesis of Horseradish Peroxidase Nanoflower@Carbon Nanotube Hybrid Nanobiocatalysts with Greatly Enhanced Catalytic Activity

Organic-inorganic hybrid nanoflowers (NFs) consisting of horseradish peroxidase (HRP) and copper II (Cu2+) are successfully synthesized with the involvement of carbon nanotubes (CNTs) by in situ and post-modification methods. Catalytic activities of in situ synthesized HRP-NF@CNT (HRP-NF@CNT-Is) and post-modification-synthesized HRP-NF@CNTs (HRP-NF@CNT-Pm) are systematically examined. The 30 mg CNTs incorporated HRP-NF@CNT-Is (HRP-NF@CNT-30Is) exhibits greatly increased catalytic activity and stability toward 3,3',5,5'-tetramethylbenzidine (TMB), thanks to the synergistic effect between HRP-NF and CNTs and the peroxidase-like activity of CNTs in the presence of hydrogen peroxide (H2O2). While HRP-NF@CNT-30Is retains almost 85% of its initial activity even after 10 cycles, HRP-NF (without CNTs) loses half of its initial activity at the same experimental conditions. We study how two experimental parameters, the pH values and temperatures, influence the catalytic activity of HRP-NF@CNT-30Is, in addition to the fact that HRP-NF@CNT-30Is is employed to detect the presence of H2O2 and glutathione (GSH) with colorimetric and spectrophotometric readouts. For instance, HRP-NF@CNT-30Is is used to sensitively detect H2O2 in the range of 20 to 300 μM with an LOD of 2.26 μM. The catalytic activity of HRP-NF@CNT-30Is is suppressed in the presence of GSH, and then an obvious color change from blue to nearly colorless is observed. Using this strategy, GSH is also sensitively determined in the range of 20-200 μM with an LOD of 11.2 μM. We expect that HRP-NF@CNTs can be used as a promising and novel nanobiocatalyst for various biomedical and industrial applications in the near future.

Octenyl succinic anhydride modified starch with excellent emulsifying properties prepared by selective hydrolysis of supramolecular immobilized enzyme

Octenyl succinic anhydride modified starch is a common green and safe emulsifier. Although the conventional pretreatment method of free enzyme hydrolysis increases the hydroxyl content on the starch surface, thus improving the grafting degree of octenyl succinic anhydride and the amphiphilicity of the modified starch, the amylose and amylopectin structures are indiscriminately hydrolyzed, reducing the emulsion stability of modified starch. In this work, α-amylase organic-inorganic hybrid nanoflower biocatalyst is designed and synthesized for pretreatment of synthetic octenyl succinic anhydride modified starch. The α-amylase organic-inorganic hybrid nanoflower biocatalyst with a unique micro-nano spatial structure can selectively hydrolyze the amylopectin and protect the amylose of starch. The amylose ratio of starch pretreated by nanoflower biocatalyst is about twice that of starch pretreated by free enzyme, reaching 22.62 %. Meanwhile, the granular structure of starch is not damaged. The obtained octenyl succinic anhydride modified starch exhibits a high degree of substitution, up to 0.0213. The emulsion prepared with this modified starch maintains excellent emulsifying properties and stability. This study provides a novel strategy for the preparation of octenyl succinic anhydride modified starch with excellent emulsifying properties, which promote the application of octenyl succinic anhydride modified starch in food, pharmaceutical and cosmetic industries.

Immobilization of Nuclease P1 Based on Hybrid Nanoflowers with Tremendously Enhanced Catalytic Activity and Stability

Organic–inorganic hybrid nanoflowers (hNFs), as a novel enzyme immobilization mode, have gained extensive attention in recent years owing to their unique high specific surface area and distinctive microstructure. For the first time, a nuclease P1(NP1)-inorganic hNF (NP1-hNF) was prepared by the self-assembly co-precipitation method using CaCl2 as the inorganic component and BSA as an inert protein. The hNF microstructure of the prepared NP1-hNFs was confirmed by scanning electron microscopy. With adding inert protein BSA, the NP1-hNFs displayed dramatically enhanced catalytic activity and improved thermal stability compared with the free enzyme. The enzyme activity yield in the immobilization process reached 266%. Comparing with the free NP1, the optimum reaction temperature of the NP1-hNFs increased 10 °C. The NP1-hNFs were further entrapped in calcium alginate microbeads to improve their reusability. The NP1-hNF microbeads retained 65% of residual activity after five recycle uses. Additionally, the microbeads retained up to 96% residual activity after 50 days of storage. This provides a promising route for enzyme immobilization in industrial application.

Synthesis of Novel Polymer-Assisted Organic-Inorganic Hybrid Nanoflowers and Their Application in Cascade Biocatalysis.

This study reports on the synthesis of novel bienzyme polymer-assisted nanoflower complexes (PANF), their morphological and structural characterization, and their effectiveness as cascade biocatalysts. First, highly porous polyamide 6 microparticles (PA6 MP) are synthesized by activated anionic polymerization in solution. Second, the PA6 MP are used as carriers for hybrid bienzyme assemblies comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). Thus, four PANF complexes with different co-localization and compartmentalization of the two enzymes are prepared. In samples NF GH/PA and NF GH@PA, both enzymes are localized within the same hybrid flowerlike organic-inorganic nanostructures (NF), the difference being in the way the PA6 MP are assembled with NF. In samples NF G/PAiH and NF G@PAiH, only GOx is located in the NF, while HRP is preliminary immobilized on PA6 MP. The morphology and the structure of the four PANF complexes have been studied by microscopy, spectroscopy, and synchrotron X-ray techniques. The catalytic activity of the four PANF was assessed by a two-step cascade reaction of glucose oxidation. The PANF complexes are up to 2-3 times more active than the free GOx/HRP dyad. They also display enhanced kinetic parameters, superior thermal stability in the 40-60 °C range, optimum performance at pH 4-6, and excellent storage stability. All PANF complexes are active for up to 6 consecutive operational cycles.