Hollow zeolite microspheres as a nest for enzymes: a new route to hybrid heterogeneous catalysts

In the field of heterogeneous catalysis, the successful integration of enzymes and inorganic catalysts could pave the way to multifunctional materials which are able to perform advanced cascade reactions. However, such combination is not straightforward, for example in the case of zeolite catalysts for which enzyme immobilization is restricted to the external surface. Herein, this challenge is overcome by developing a new kind of hybrid catalyst based on hollow zeolite microspheres obtained by the aerosol-assisted assembly of zeolite nanocrystals. The latter spheres possess open entry-ways for enzymes, which are then loaded and cross-linked to form cross-linked enzyme aggregates (CLEAs), securing their entrapment. This controlled design allows the combination of all the decisive features of the zeolite with a high enzyme loading. A chemo-enzymatic reaction is demonstrated, where the structured zeolite material is used both as a nest for the enzyme and as an efficient inorganic catalyst. Glucose oxidase (GOx) ensures the in situ production of H₂O₂ subsequently utilized by the TS-1 zeolite to catalyze the epoxidation of allylic alcohol toward glycidol. The strategy can also be used to entrap other enzymes or combination of enzymes, as demonstrated here with combi-CLEAs of horseradish peroxidase (HRP) and glucose oxidase. We anticipate that this strategy will open up new perspectives, leveraging on the spray-drying (aerosol) technique to shape microparticles from various nano-building blocks and on the entrapment of biological macromolecules to obtain new multifunctional hybrid microstructures.

A spray drying technique is used to prepare hollow zeolite microparticles into which an enzyme can be entrapped. Via this “Lego-like” strategy, we create hybrid heterogeneous catalysts that can run multistep chemo-enzymatic cascade reactions.     

Enzyme-modified nanoparticles using biomimetically synthesized silica

The entrapment of enzymes within biomimetic silica nanoparticles offers unique and simple immobilization protocols that merge the stability of proteins confined in solid phases with the high loading and reduced diffusion limitations inherent to nano-sized structures. Herein, we report on the biomimetic silica entrapment of chemically derivatized horseradish peroxidase for amperometric sensing applications. Scanning electron microscopy shows evidence of the formation of enzyme-modified nanospheres using poly(ethylenimine) as a template for silicic acid condensation. When these nanospheres are directly deposited on graphite electrodes, chemically modified anionic peroxidase shows direct electron transfer at 0 mV vs Ag|AgCl. Microgravimetric measurements as well as SEM images demonstrate that negatively charged peroxidase is also entrapped when silica precipitates at gold electrodes are modified with a self-assembled monolayer of poly(ethylenimine). Electrostatic interactions may play a crucial role for efficient enzyme entrapment and silica condensation at the PEI template monolayer. The in-situ biomimetically synthesized peroxidase nanospheres are catalytically active, enabling direct bioelectrocatalysis at 0 mV vs Ag|AgCl with long-term stability.     

Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures

Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete glucose oxidase (GOx)/horseradish peroxidase (HRP) enzyme pairs on specific DNA origami tiles with controlled interenzyme spacing and position. The distance between enzymes was systematically varied from 10 to 65 nm, and the corresponding activities were evaluated. The study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally limited diffusion of intermediates across connected protein surfaces contributed to the enhancement in activity for closely spaced GOx/HRP assemblies. To further test the role of limited dimensional diffusion along protein surfaces, a noncatalytic protein bridge was inserted between GOx and HRP to connect their hydration shells. This resulted in substantially enhanced activity of the enzyme pair.     

A Neural Network Model in the Calibration of Glucose Sensor Based on the Immobilization of Glucose Oxidase into Polypyrrole Matrix

The immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power.     

Nanocomposites of nanocrystalline cellulose for enzyme immobilization

We describe the synthesis, characterization and use of a composite material made of a renewable source and metallic nanoparticles for biosensing applications. Nanocrystalline cellulose (NCC) is a product isolated from natural cellulose fibers, which is of approximately 100 nm long and 10 nm wide in size. We augmented the surface area and tailored the chemical affinity of NCC by optimally dressing it with gold nanoparticles (AuNPs). The deposition of AuNPs on NCC was controlled by using cationic polyethylenimine (PEI) at different pHs. AuNPs were thiol-functionalized using different linkers prior to enzyme immobilization. The enzyme (glucose oxidase or GOx) was conjugated on the composite by carbodiimide coupling, and subsequent activation of linker-carboxylic acid group. Our results showed that GOx was attached to the surface of the NCC nanocomposite. Moreover, the amount of GOx loaded onto the support depended on the length of the thiol-linker used. The lower value (20.3 mg/mg of support) was obtained with the longer thiol-linker (11 carbon chain) compared to 25.2 mg/mg of support for the smaller thiol-linker (3 carbon chain).     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

A Three-Enzyme Cascade Reaction through Positional Assembly of Enzymes in a Polymersome Nanoreactor

Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS^.+. Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.     

生物/化学同步聚合法制备纤维蛋白纳米复合物及传感研究

本文以有效提高生物分子包埋率为目的,基于生物/化学同步聚合的新方法制备了一种新型纤维蛋白聚合物基纳米复合物,并研究了该复合物修饰电极的传感性能。该方法在凝血仿生聚合的同时,采用NaAuCl4作为氧化剂化学氧化聚合生成聚多巴胺(PDA),在PDA膜内原位合成纳米金(AuNPs),同时在PDA-纤维蛋白凝胶生长时包埋葡萄糖氧化酶(GOx)。生物/化学同步聚合法操作简单,条件温和。该纳米复合物引入了AuNPs的优异性质,有效提升了GOx的包埋量,所制电化学生物传感器对葡萄糖的检测灵敏度高达117μA/(cm2·mmol/L),检测限为57nmol/L。     

Preserving the activity of enzymes under harsh oxidizing conditions: sol–gel entrapped alkaline phosphatase exposed to bromine

Chemically reactive sol–gel matrices hold the ability of protecting entrapped enzymes from destruction by external harsh chemicals. We show this concept by exposing alkaline phosphatase (AlP) to a strong oxidizing agent—bromine. In solution, AlP is immediately destroyed by this oxidant. When AlP was entrapped in hybrid silica sol–gel materials carrying double bonds, the reactivity of AlP was preserved after exposure to bromine under conditions which totally destroy it in solution. The matrices studied were vinylated and allylated silicas, and their protectability was compared to n-alkylated silicas and to silica itself. For instance, the reactivity of AlP entrapped in allylated silica after exposure to 25.6 mM bromine solution is 40 times higher than its reactivity when entrapped in pure silica; and in solution the enzyme is totally destroyed at this concentration. Molecular level mechanisms for these observations are proposed.     

Fabrication of Bienzymatic Glucose Biosensor Based on Novel Gold Nanoparticles‐Bacteria Cellulose Nanofibers Nanocomposite

An exploration of gold nanoparticles–bacterial cellulose nanofibers (Au‐BC) nanocomposite as a platform for amperometric determination of glucose is presented. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) were immobilized in Au‐BC nanocomposite modified glassy carbon electrode at the same time. A sensitive and fast amperometric response to glucose was observed in the presence of electron mediator (HQ). Both of GOx and HRP kept their biocatalytic activities very well in Au‐BC nanocomposite. The detection limit for glucose in optimized conditions was as low as 2.3 µM with a linear range from 10 µM to 400 µM. The biosensor was successfully applied to the determination of glucose in human blood samples.     

Hydrogen Peroxide Sensor Based on Horseradish Peroxidase Combined with CaCO3 Microspheres and Gold Nanoparticles

Direct electrochemistry and electrocatalysis of horseradish peroxidase(HRP) were achieved by entrapping the enzyme between CaCO3 microspheres and gold nanoparticles through forming sandwich configuration (CaCO3-HRP-AuNPs). Polyanion, poly(styrene sulfonate)(PSS), was hybrid with CaCO3 microspheres to increase the surface negative charges for binding with HRP through electrostatic interaction. After the bioconjugate CaCO3 PSS-HRP was entrapped in chitosan based sol-gel(CS-GPTMS) film, HRP was encapsulated by in situ formation of an outer layer of AuNPs through electrochemical reduction of HAuCl4. The composite film containing AuNPs, CaCO3-PSS-HRP bioconjugates and CS-GPTMS can provide favorable microenvironment for HRP to perform direct electron transfer at glassy carbon electrode(GCE). HRP retained its bioelectrocatalytic activity and lead to sensitive and fast amperometric response for the determination of H2O2. H2O2 could be detected in a very wide linear range from 5.0×10–6 mol/L to 7.1×10–2 mol/L. The sandwich configuration of CaCO3-biomolecules-AuNPs could serve as a versatile platform for enzyme immobilization and biosensing.     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.     

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile,Uniform, Enzyme-Loaded Organic Nanoparticles

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5–12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.     

Erratum to: Preserving the activity of enzymes under harsh oxidizing conditions: sol–gel entrapped alkaline phosphatase exposed to bromine

Chemically reactive sol–gel matrices hold the ability of protecting entrapped enzymes from destruction by external harsh chemicals. We show this concept by exposing alkaline phosphatase (AlP) to a strong oxidizing agent—bromine. In solution, AlP is immediately destroyed by this oxidant. When AlP was entrapped in hybrid silica sol–gel materials carrying double bonds, the reactivity of AlP was preserved after exposure to bromine under conditions which totally destroy it in solution. The matrices studied were vinylated and allylated silicas, and their protectability was compared to n-alkylated silicas and to silica itself. For instance, the reactivity of AlP entrapped in allylated silica after exposure to 25.6 mM bromine solution is 40 times higher than its reactivity when entrapped in pure silica; and in solution the enzyme is totally destroyed at this concentration. Molecular level mechanisms for these observations are proposed.     

Bioinorganic Nanocomposite Hydrogels Formed by HRP–GOx‐Cascade‐Catalyzed Polymerization and Exfoliation of the Layered Composites

The mild preparation of multifunctional nanocomposite hydrogels is of great importance for practical applications. We report that bioinorganic nanocomposite hydrogels, with calcium niobate nanosheets as cross‐linkers, can be prepared by dual‐enzyme‐triggered polymerization and exfoliation of the layered composite. The layered HRP/calcium niobate composites (HRP=horseradish peroxidase) are formed by the assembly of the calcium niobate nanosheets with HRP. The dual‐enzyme‐triggered polymerization can induce the subsequent exfoliation of the layered composite and final gelation through the interaction between polymer chains and inorganic nanosheets. The self‐immobilized HRP‐GOx enzymes (GOx=glucose oxidase) within the nanocomposite hydrogel retain most of enzymatic activity. Evidently, their thermal stability and reusability can be improved. Notably, our strategy could be easily extended to other inorganic layered materials for the fabrication of other functional nanocomposite hydrogels.     

Effect of Gold Nanoparticles on the Structure and Electron‐Transfer Characteristics of Glucose Oxidase Redox Polyelectrolyte‐Surfactant Complexes

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox‐active polyelectrolyte–surfactant complex containing [Os(bpy)₂Clpy]²⁺ (bpy=2,2′‐bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron‐transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing‐incidence small‐angle X‐ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz‐crystal microbalance with dissipation (QCM‐D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron‐transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five‐fold increase in current response to glucose compared with analogous supramolecular AuNP‐free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron‐transfer process.     

Nanocomposite Incorporating V2O5 Nanowires and Gold Nanoparticles for Mimicking an Enzyme Cascade Reaction and Its Application in the Detection of Biomolecules

Artificial enzyme mimics are a current research interest, and many nanomaterials have been found to display enzyme‐mimicking activity. However, to the best of our knowledge, there have not hitherto been any reports on the use of pure nanomaterials to construct a system capable of mimicking an enzyme cascade reaction. Herein, we describe the construction of a novel nanocomposite consisting of V₂O₅ nanowires and gold nanoparticles (AuNPs) through a simple and facile chemical method, in which V₂O₅ and AuNPs possess intrinsic peroxidase and glucose oxidase (GOx)‐like activity, respectively. Results suggest that this material can mimic the enzyme cascade reaction of horseradish peroxidase (HRP) and GOx. Based on this mechanism, a direct and selective colorimetric method for the detection of glucose has been successfully designed. Because single‐strand and double‐strand DNA (ssDNA and dsDNA) have different deactivating effects on the GOx‐like activity of AuNPs, the sensing of target complementary DNA can also be realized and disease‐associated single‐nucleotide polymorphism of DNA can be easily distinguished. Our study opens a new avenue for the use of nanomaterials in enzyme mimetics, and holds promise for the further exploration of nanomaterials in creating alternative catalytic systems to natural enzymes.     

Carbon Cavity Microelectrode for Electrical Wiring of Enzyme by Insoluble Electroactive Species in Aqueous Media

The carbon cavity microelectrode (CME), exhibiting a volume of 4×10^?6 cm³, offers a genuine alternative for immobilizing and connecting enzymes in aqueous electrolytes by powder of insoluble redox materials. In the present work, the electrochemical behavior of two redox species such as ferrocene (Fc) and tetrathiafulvalene (TTF) was investigated with CME to evaluate their potentialities in the electrical wiring of enzymes. For this purpose, powder of two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), was independently mixed with an insoluble redox material and forced to fill the single micro cavity of a carbon electrode covered by an inert insulator. The presence of the electroactive species, as well as the enzyme wiring was investigated by cyclic voltammetry. The amperometric detection of glucose was carried out by potentiostating the TTF/GOx and the Fc/GOx microelectrodes at 0.25 and 0.35 V respectively. The amperometric detection of H₂O₂ by the TTF/HRP microelectrode was performed at ?0.1 V vs. SCE.     

Acetylcholinesterase Immobilization on Polyacrylamide/Functionalized Multi-walled Carbon Nanotube Nanocomposite Nanofibrous Membrane

In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers’ morphology and conductivity) and chemical (enzyme entrapment) methods.

Activity of AC electrokinetically immobilized horseradish peroxidase

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H₂O₂ to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.