Organic-inorganic Hybrid Nanoflowers Research Articles

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.

Immobilization of Bacillus amyloliquefaciens protease “Neutrase” as hybrid enzyme inorganic nanoflower particles: A new biocatalyst for aldol-type and multicomponent reactions

Organic–inorganic hybrid nanoflowers (hNFs) with commercial protease “Neutrase” is proposed and characterized as efficient and green biocatalysts for promiscuous catalysis in aldol-type and multicomponent reactions. Neutrase hNFs [Neutrase-(Cu/Ca/Co/Mn)3(PO4)2] are straightforwardly prepared through mixing metal ion (Cu2+, Ca2+, Co2+ or Mn2+) aqueous solutions with Neutrase in phosphate buffer (pH 7.4, 10 mM) resulting in precipitation (3 days). The hNFs were characterized by various techniques including scanning electron microscopy (SEM), energy dispersive X-ray (EDX), element mapping, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). In SEM images, the metal-Neutrase complexes revealed flower-like or granular structures after hybridization. The effect of metal ions and enzyme concentrations on the morphology and enzyme activity of the Neutrase-hNFs was examined. The synthesized Neutrase-Mn hNFs showed superior activity and stability compared to free Neutrase. Traditional organic CC coupling reactions such as aldol condensation, decarboxylative aldol, Knoevenagel, Hantzsch-type reactions and synthesis of 4H-pyran derivatives were used to test the generality and scope of Neutrase promiscuity, while optimizing conditions for the Neutrase-Mn hNF biocatalyst. Briefly, Neutrase-Mn3(PO4)2 hNFs showed excellent enzyme activity, stability and reusability, qualifying as effective reusable catalysts for coupling reactions under mild conditions.

Synthesis of Enzyme-based Organic-Inorganic Hybrid Nanoflower Particles

Enzyme-incorporated hybrid nanostructures are the immobilization of enzymes and inorganic components that exhibits promising characteristics in various industries. The immobilization of enzymes onto nanomaterial is naturally based to accommodate the enzymatic activity, stability, recyclability as well as their catalytic functions. The designing of these conjugates can improve the overall enzymatic performance by imparting their novel properties onto the system in comparison to conventional free enzymes which experience drawbacks in terms of deactivation or denaturing. A facile and ultrafast method is described in this paper to synthesize a novel enzyme-incorporated lipase/Cu 3 (PO 4 ) 2 hybrid nanoflower (NF). The physical properties of the hybrid NF allow easier retrieval which indicates its higher reusability and recyclability value. The enzyme loading capacity was found to be 95.1% whereas, the catalytic performance of lipase/Cu 3 (PO 4 ) 2 hybrid NF at the optimal conditions resulted in a specific enzyme activity of 1752 U/g corresponding to an increment of 90.5% to that of free lipase. This indicates that the well-designed lipase/Cu 3 (PO 4 ) 2 hybrid NF to be highly efficient in industrial biocatalytic applications. Meanwhile, in future work, we aim to study its operational stability and reusability to enzymatically degrade biopolymers through hydrolysis process.

Synthesis of organic-inorganic hybrid nanoflowers of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL): Improvement of thermal stability and reusability

Enzyme immobilization is used to improve the application of enzymes, allowing the reuse of biocatalysts and increasing their stability under reaction conditions. Immobilization of enzymes through structures, such as nanoflowers, is an innovative, simple, and low-cost method compared to other techniques. In this context, the main objective of this work is to synthesize hybrid biocatalytic nanostructures, similar to flowers, of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL). The production of nanoflowers occurred by precipitation of lipases with CuCl2 or CuSO4 salts for 72 h. However, challenges and obstacles were faced in obtaining effective and practical nanoflowers, such as nanoflowers’ low thermal stability and reusability. To overcome these challenges, two conditions were tested: nanoflowers cross-linked with glutaraldehyde and nanoflowers and nanoparticles cross-linked with glutaraldehyde. This last biocatalyst prepared by CuSO4 precipitation showed better thermal stability (half-life about 230 and 233 min for CALB and TLL, respectively, under incubation at 60 °C and pH 7). The CALB biocatalyst retained 70 % of its initial activity (2.31 U) after 10 cycles of hydrolysis. Therefore, this work shows not only the problems and barriers of nanoflowers synthesis, but also the possibility of producing more stable and efficient biocatalysts using improved protocols.

Synthesis and Characterization of Copper-Nanoflowers with the Utilization of Medicinal Plant Extracts for Enhanced Various Enzyme Inhibitory Activities.

In this study, organic-inorganic hybrid nanoflowers were synthesized using methanolic extracts of the medicinal plants Ajuga chamaepitys subsp. chia var. chia, Achillea wilhelmsii, Bongardia chrysogonum, Malva sylvestris, Phlomis grandiflora var. grandiflora, Verbascum sp. together with copper ions (Cu2+ ). The synthesized plant extract based-inorganic hybrid nanoflowers (PE-ihNFs) of A. chamaepitys subsp. chia var. chia (Ac-ihNFs), A. wilhelmsii (Aw-ihNfs), B. chrysogonum (Bc-ihNFs), M. sylvestris (Ms-ihNFs) P. grandiflora var. grandiflora (Pg-ihNFs), and Verbascum sp. (Vs-ihNFs) were characterized by Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray (EDX), Fourier transform infrared spectrometry (FTIR), and X-Ray Diffraction (XRD). Also, several enzymes were selected to evaluate the enzyme inhibition activities of the synthesized PE-ihNFs. For the first-time, enzymes, tyrosinase, α-amylase and α-glucosidase, acetyl and butyryl cholinesterase inhibition activities of the PE-ihNFs with comparison to their plain plant extracts were evaluated in vitro. Results show that the among all the analyzed PE-ihNFs, demonstrated better α-glucosidase & α-amylase enzyme inhibition activity compared to the plain extracts. These initial studies are promising for the synthesis of these hybrid nanoflowers containing medicinal plant extracts, which might have commercial applications in the pharmaceutical and dermo-cosmetics industries.

Fabrication of myoglobin hybrid nanoflowers for decolorization process of evans blue and congo red

• Synthesis and characterization of Myoglobin hybrid nanoflowers (MbNFs@Cu) in the first time. • Enhanced peroxidase-like activities of MbNFs@Cu. • The MbNFs@Cu exhibited excellent dye decolorization efficiency for Evans blue and Congo red dyes. Here, myoglobin hybrid nanoflowers (MbNFs@Cu) were synthesized in various pH values and protein concentrations considering the organic–inorganic hybrid nanoflower method for the first time. Synthesized material was characterized by using EDX, XRD, and FTIR analyses. After determining the optimum morphology, the MbNFs@Cu exhibited great stability and peroxidase-mimic activity towards various pH values and temperature, and provided excellent dye decolorization efficiency for evans blue (EB) and congo red (CR) dyes with more than 90 % within 20 min. These results prove that this material has a potential in the removal of some organic dyes and in catalytic applications.

Chitosan-regulated biomimetic hybrid nanoflower for efficiently immobilizing enzymes to enhance stability and by-product tolerance

Organic-inorganic hybrid nano-materials have been considered to be promising immobilization matrixes for enzymes due to their significantly enhanced reusability and stability of enzymes. Herein, we constructed a novel organic-inorganic hybrid nanoflower via biomacromolecule-regulated biomimetic mineralization to immobilize sucrose phosphorylase (SPase). It was found that chitosan (CS) effectively regulated the biomimetic mineralization of calcium phosphate (CaP), leading to the formation of flower-like hybrid materials for the entrapment of SPase via self-assembly to establish a nano-biocatalyst (CS-CaP@SPase). Upon immobilization, the obtained CS-CaP@SPase exhibited excellent pH, by-product and organic solvents tolerance, and storage stability. Specifically, at acidic condition (pH 4), CS-CaP@SPase performed over 80 % of initial activity, which was 2.42-folds higher than that of free SPase. The catalytic activity of free SPase was severely inhibited about 30 % in the presence of fructose (1.2 M), but CS-CaP@SPase only lost 5 % relative activity. The CS-CaP@SPase retained over 80 % of its relative activity, while the free SPase maintained <20 % of its relative activity in acetonitrile. The relative activity of CS-CaP@SPase was still retained about 80 % after 10 cycles and maintained 75 % after 15 days. Based on Raman spectra analysis, it was also found that the increased β-folding component of SPase in the secondary structure after immobilization was the main factor for its enhanced stability. It is reasonable to believe that biomacromolecule-regulated biomimetic mineralization could be potentially used as a promising method to immobilize enzymes with excellent stability and recyclability, thereby facilitating the preparation of highly efficient catalysts for industrial biocatalysts, biosensing, and biomedicine.

Bio-hybrid nanoarchitectonics of nanoflower-based ELISA method for the detection of Staphylococcus aureus

Staphylococcus aureus is one of the crucial bacterial pathogens that cause food poisoning and human infectious diseases. Therefore, the establishment of a rapid and sensitive detection method for S. aureus is of great significance for the diagnosis, prevention, and control of the diseases caused by this pathogen. To this end, we have established a bio-hybrid nanoarchitectonics of nanoflower-based ELISA method for the detection of S. aureus with high specificity and sensitivity. The core of the method is to prepare a specific cell wall binding domain (CBD)-horseradish peroxidase (HRP)-Cu3(PO4)2 three-in-one organic-inorganic hybrid nanoflower complex through nanoarchitectonics. Detection consists of two main steps: we firstly coated an anti-S. aureus polyclonal antibody into the wells of a 96-well plate to specifically capture S. aureus, and then added customized CBD-HRP-Cu3(PO4)2 organic-inorganic hybrid nanoflower to the system. Due to the high specificity of the CBD from lysin PlyV12 of bacteriophage ϕ1 with S. aureus, a stable antibody-bacterium-hybrid nanoflower complex was formed. Then, the signal amplification of HRP enzyme activity in the hybrid nanoflower was carried out by using the TMB (3,3′,5,5′-tetramethylbenzidine)-hydrogen peroxide reporting system. The method has been proved to be capable of specifically detecting S. aureus ranging from 101 to 106 conoly-forming unit (CFU)/mL linearly with a detection limit down to 6 CFU/mL, which is arguably one of the best for all detection methods reported to date.