Efficient Enzyme Immobilization Research Articles

Sensitive enzymatic glucose biosensor fabricated by electrospinning composite nanofibers and electrodepositing Prussian blue film

In this study, a simple, novel method of preparing glucose amperometric biosensors is reported. This biosensor is based on the quantitative measurement of an intermediate product, hydrogen peroxide (H2O2), which in turn is oxidized by Prussian blue film in composite nanofibers. The biocomposite is composed of Prussian blue, chitosan, and polyvinyl alcohol fabricated by electrodeposition and subsequent electrospinning in enzyme-friendly conditions. The resulting biocomposite nanofibers with porous structures and good biocompatibility sustained the stability of the Prussian blue film and the efficient enzyme immobilization without additional cross-linking agents. The stability of the Prussian blue film at neutral and weak alkalescent solutions was increased after the modification. Furthermore, glucose oxidase retained the biocatalytic activities and glucose is oxidized by dissolved oxygen efficiently, which is catalyzed by glucose oxidase to produce hydrogen peroxide. The exhibited good linear behavior in glucose concentrations ranging from 3.30×10−6M to 5.56×10−2M with a low detection limit of 3.61×10−7M. Moreover, the fabricated biosensor exhibited long-term stability, good reproducibility, and absence of interference from other co-existing electroactive species. Thus, the facile and effective methodology of sensor preparation in this study will promote further electrochemical research on proteins, biosensors, and other bioelectrochemical devices.

Bioactive flake-shell capsules: soft silica nanoparticles for efficient enzyme immobilization.

Amorphous silica particles are used extensively in industrial processes as well as in scientific and biomedical research. Flake-shell silica nanoparticles, prepared by self-templating hydrothermal synthesis, exhibit large surface areas and pore volumes, hollow spherical morphology, as well as hydrophilic surfaces, affording great potential for their application in enzyme immobilization. In this work we show that the shell, which is composed of a network of silica flakes, promotes uptake of enzymes of different sizes, i.e., lysozyme, lipase, and chymotrypsin. Moreover, the capsules provide a favorable porous structure for diffusion of small substrate molecules while permitting enzymes to retain their activity. The silica composition of the capsules provides structural stability, biocompatibility and allows easy functionalization. Amine and dextran functionalities were introduced to modify the shell morphology and to control enzyme loading and activity. The efficient protein immobilization on these inorganic capsules should lead to their application in biocatalysis, biosensing and drug delivery.

Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres

In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500–600nm) were synthesized by a modified sol–gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100–150mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4mM, 13.36mM/min) were close to those of the free CAT (25.7mM, 13.46mM/min).

Metal–Organic Coordination-Enabled Layer-by-Layer Self-Assembly to Prepare Hybrid Microcapsules for Efficient Enzyme Immobilization

A novel layer-by-layer self-assembly approach enabled by metal-organic coordination was developed to prepare polymer-inorganic hybrid microcapsules. Alginate was first activated via N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) coupling chemistry, and subsequently reacted with dopamine. Afterward, the dopamine modified alginate (Alg-DA) and titanium(IV) bis(ammonium lactato) dihydroxide (Ti(IV)) were alternatively deposited onto CaCO3 templates. The coordination reaction between the catechol groups of Alg-DA and the Ti(IV) allowed the alternative assembly to form a series of multilayers. After removing the templates, the alginate-titanium hybrid microcapsules were obtained. The high mechanical stability of hybrid microcapsules was demonstrated by osmotic pressure experiment. Furthermore, the hybrid microcapsules displayed superior thermal stability due to Ti(IV) coordination. Catalase (CAT) was used as model enzyme, either encapsulated inside or covalently attached on the surface of the resultant microcapsules. No CAT leakage from the microcapsules was detected after incubation for 48 h. The encapsulated CAT, with a loading capacity of 450-500 mg g(-1) microcapsules, exhibited desirable long-term storage stability, whereas the covalently attached CAT, with a loading capacity of 100-150 mg g(-1) microcapsules, showed desirable operational stability.

A novel amperometric hydrogen peroxide biosensor based on pyrrole-PAMAM dendrimer modified gold electrode

Horseradish peroxidase (HRP) was immobilized into an electrochemically prepared copolymer of pyrrole–PAMAM (PAMAM; polyamidoamine) dendrimers for the construction of amperometric hydrogen peroxide biosensor. First, second, and third generation amidoamine–pyrrole dendrons having branched amine periphery and focal pyrrole functionality were synthesized via divergent pathway. Pyrrole dendrimers were covalently attached onto the electrode surface and polymerized by electrochemical copolymerization with pyrrole monomer. The synthesized dendrimers and copolymers have been characterized by FTIR-ATR and NMR. These copolymers have been utilized as conducting films for amperometric hydrogen peroxide sensing. The HRP retains its bioactivity after immobilization into the dendronized pyrrole-copolymers. Amperometric response was measured as a function of concentration of hydrogen peroxide, at fixed potential of +0.35V vs. Ag/AgCl in a phosphate buffered saline (pH 7.5). The effect of pH and temperature of the medium, storage, and reusability properties were investigated. The results indicate an efficient immobilization of enzyme onto the PAMAM type dendrimer modified surface containing pyrrole monomer, which leads to high enzyme loading, and increased lifetime stability of the electrode.

Highly Efficient Enzyme Immobilization and Stabilization within Meso-Structured Onion-Like Silica for Biodiesel Production

Meso-structured onion-like silica (Meso-Onion-S) was synthesized and used as a host of enzyme immobilization. Meso-Onion-S has a 200–300 nm sized primary meso-structured onion building unit, and each onion unit has highly curved mesopores of 10 nm diameter in a multishell structure. Nanoscale enzyme reactors (NERs) in Meso-Onion-S were prepared via a two-step process of enzyme adsorption and subsequent enzyme cross-linking, which effectively prevents the leaching of cross-linked enzyme aggregates from highly curved mesopores of Meso-Onion-S. As a result, NERs in Meso-Onion-S significantly improved the enzyme stability as well as the enzyme loading. For example, NER of lipase (NER-LP) was stable under rigorous shaking for 40 days, while the control sample of adsorbed LP (ADS-LP) with no enzyme cross-linking showed a rapid inactivation due to rigorous enzyme leaching under shaking. Stable NER-LP was successfully employed to produce biodiesels and fatty acid methyl esters, from the LP-catalyzed transesterification of soybean oil with methanol. Interestingly, the specific activity of NER-LP was 23 and 10 times higher than those of free LP and ADS-LP, respectively, revealing the importance of LP stabilization in the form of NER-LP in the presence of organic solvents.

Effects of carbon structure orientation on the performance of glucose sensors fabricated from electrospun carbon fibers

High-performance enzyme-based glucose sensors were prepared by electrospinning carbon fibers. The efficiency of the glucose sensor was assessed based on efficient enzyme immobilization and electrical resistance transfer by examining improved porosity and electrical properties, respectively. The porosity of the electrospun carbon fiber electrode was improved by physical activation to increase the immobilization sites of the glucose oxidase enzyme. The electrical properties were improved by a thermal treatment, which caused carbon orientation effects because of the high thermal energy. The glucose oxidase enzyme immobilization was developed based on improved specific surface area and pore volume, which were studied by pore structure and image analyzers. The glucose sensor was evaluated by amperometric measurements and cyclic voltammetry. The measured current increased with higher glucose concentrations based on the effects of the developed pore structure and the electrical properties. The enzymatic kinetics were also studied using the Lineweaver–Burk equation. The sensitivity of the glucose sensor was improved significantly with increased maximum current, whereas the GOD enzyme activity was diminished by efficient GOD immobilization. It is concluded that a high-performance glucose sensor was obtained using electrospun carbon fibers based on the effects of efficient GOD enzyme immobilization and electrical resistance transfer.

Fabrication of a chitosan/glucose oxidase–poly(anilineboronic acid)–Aunano/Au-plated Au electrode for biosensor and biofuel cell

Enzyme immobilization is one of the key factors in constructing high-performance enzyme biosensors and biofuel cells (BFCs). Herein, we propose a new protocol for efficient immobilization of a glycoprotein enzyme based on the interaction of the 1, 2- or 1, 3-diols in the glycoprotein with a boronic acid functionalized monomer. Briefly, casting a mixture of glucose oxidase (GOx) and anilineboronic acid (ABA) followed by a NaAuCl4 solution to an Au-plated Au electrode surface yielded a GOx–poly(ABA) (PABA)–gold nanoparticle (Aunano) bionanocomposite, and chitosan (CS) was then cast and air-dried. In the present protocol, the small-sized Aunano or Au subnanostructures can form near/on the enzyme molecule, which greatly promotes the electron transfer of enzymatic reaction and enhances the amperometric responses. The thus-prepared CS/GOx–PABA–Aunano/Au-plated Au electrode worked well in the first-/second generation biosensing modes and as a bioanode in a monopolar biofuel cell, with analytical or cell-power performance superior to those of most analogues hitherto reported.

Assembly of Graphene Oxide–Enzyme Conjugates through Hydrophobic Interaction

Biochemical and biomedical applications of graphene oxide (GO) critically rely on the interaction of biomolecules with it. It has been previously reported that the biological activity of the GO-enzyme conjugate decreases due to electrostatic interaction between the enzymes and GO. Herein, the immobilization of horseradish peroxidase (HRP) and oxalate oxidase (OxOx) on chemically reduced graphene oxide (CRGO) are reported. The enzymes can be adsorbed onto CRGO directly with a tenfold higher enzyme loading than that on GO, and maximum enzyme loadings reach 1.3 and 12 mg mg(-1) for HRP and OxOx, respectively. Significantly, the more CRGO is reduced, the higher the enzyme loading. The CRGO-HRP conjugates also exhibit higher enzyme activity and stability than GO-HRP. Excellent properties of the CRGO-enzyme conjugates are attributed to hydrophobic interaction between the enzymes and the CRGO. The hydrophobic interaction mode of the CRGO-enzyme conjugates can be applied to other hydrophobic proteins, and thus could dramatically improve the performance of immobilized proteins. The results indicate that CRGO is a potential substrate for efficient enzyme immobilization, and is an ideal candidate as a macromolecule carrier and biosensor.

Surface modification of electrospun spherical activated carbon for a high-performance biosensor electrode

A glucose sensor electrode was prepared from electrospun spherical-type carbon materials. The glucose oxidase (GOD) enzyme was immobilized on a prepared electrode for efficient glucose sensing. The GOD immobilization was maximized by enlarged sites of carbon electrode and improved interfacial affinity between the carbon surface and the GOD achieved via physical activation and oxyfluorination, respectively. The specific surface area was enlarged significantly, by over 42 fold, through physical activation. In addition, the hydrophobic carbon surface was modified with hydrophilic functional groups by direct oxyfluorination for improved interfacial affinity between the carbon and GOD. Accordingly, the GOD immobilization improved significantly by approximately 9 fold. The glucose sensor was evaluated by amperometric measurements and cyclic voltammetry. The measured current increased with higher glucose concentrations based on the effects of the developed pore structure and the introduced hydrophilic functional groups. The enzymatic kinetics were also studied using the Lineweaver–Burk equation. The sensitivity of the glucose sensor was improved by approximately 3 fold with increased maximum current, whereas the GOD enzyme activity was diminished by efficient GOD immobilization. Conclusively, a high-performance glucose sensor was obtained using an electrospun spherical-type carbon material due to efficient GOD enzyme immobilization.

Shape reformable polymeric nanofibers entrapped with QDs as a scaffold for enzyme stabilization

In this study, we report the structure and shape of new polymeric nanofibers entrapped with QDs, which were prepared by electrospinning a homogenous mixture of polymers and QDs. Uniformly distributed QDs within the polymer matrix seem to induce the high degree of compactness and shape rigidity in the nanofibers, and resulted in the efficient enzyme immobilization. After immobilization, the esterase enzymes coated on the QDs–nanofibers showed good stability with no loss of enzyme activity even after being recycled for more than ten times. The present study demonstrates the successful application of shape reformable polymeric nanofibers entrapped with QDs for enzyme immobilization for the first time, and so it is anticipated that this technology can be employed in various enzyme applications due to its facile shape control feature in the fabrication of the nanofibers.

Glucose Biosensor Based on the Fabrication of Glucose Oxidase in the Bio‐Inspired Polydopamine‐Gold Nanoparticle Composite Film

AbstractA highly efficient enzyme immobilization method has been developed for electrochemical biosensors using polydopamine films with gold nanoparticles (AuNPs) embedded. This simple enzyme fabrication method can be performed in very mild conditions and stored in a long time with high bioactivity. The fabricated amperometric glucose biosensor exhibited a high and reproducible sensitivity, wide linear dynamic range and low limit of detection (LOD) (0.1 μmol·L−1). A low value of 1.5 mmol·L−1 for the apparent Michaelis‐Menten constant KappM was obtained. The high sensitivity, wide linear range, good reproducibility and stability make this biosensor a promising candidate for portable amperometric glucose biosensor.

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

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.

Enzymes in food processing: a condensed overview on strategies for better biocatalysts.

Food and feed is possibly the area where processing anchored in biological agents has the deepest roots. Despite this, process improvement or design and implementation of novel approaches has been consistently performed, and more so in recent years, where significant advances in enzyme engineering and biocatalyst design have fastened the pace of such developments. This paper aims to provide an updated and succinct overview on the applications of enzymes in the food sector, and of progresses made, namely, within the scope of tapping for more efficient biocatalysts, through screening, structural modification, and immobilization of enzymes. Targeted improvements aim at enzymes with enhanced thermal and operational stability, improved specific activity, modification of pH-activity profiles, and increased product specificity, among others. This has been mostly achieved through protein engineering and enzyme immobilization, along with improvements in screening. The latter has been considerably improved due to the implementation of high-throughput techniques, and due to developments in protein expression and microbial cell culture. Expanding screening to relatively unexplored environments (marine, temperature extreme environments) has also contributed to the identification and development of more efficient biocatalysts. Technological aspects are considered, but economic aspects are also briefly addressed.

Efficient and stable enzyme immobilization in a block copolypeptide vesicle-templated biomimetic silica support

We report the immobilization of a model enzyme, papain, within silica matrices by combining vesiclization of poly- l-lysine-b-polyglycine block copolypeptides with following silica mineralization. Our novel strategy utilizes block polypeptide vesicles to induce the condensation of orthosilicic acid while trapping an enzyme within and between vesicles. The polypeptide mediated silica-immobilized enzyme exhibits enhanced pH and thermal stability and reusability, comparing with the free and vesicle encapsulated enzyme. The enhanced enzymatic activity in the immobilized enzyme is due to the confinement of the enzyme in the polypeptide mediated silica matrices. Kinetic analysis shows that the enzyme functionality is determined by the structure and property of silica/polypeptide matrices. The proposed novel strategy provides an alternative route for the synthesis of a broad range of functional bionanocomposites entrapped within silica nanostructures.

Effective immobilization of enzyme in glycidoxypropyl-functionalized periodic mesoporous organosilicas (PMOs)

Bifunctional periodic mesoporous organosilicas (PMOs) with ethane bridging groups within the framework and various amounts of terminally bonded groups in the pore channels was synthesized by the co-condensation of 1,2-bis(triethoxysilyl)ethane (BTESE) and 3-glycidoxypropyltrimethoxylsilane (GPTMS) in the presence of triblock copolymer poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (P123) surfactants under acidic conditions and utilized as supports for enzyme immobilization. It is revealed that glycidoxypropyl groups have been successfully covalently attached to the pore wall of PMOs and a fraction of them have experienced epoxy ring opening reaction to form diol groups. The functional materials still preserve a mesoscopic ordering at a concentration of GPTMS as high as 10% in the reaction mixtures. The BET surface area, pore volume and pore size of the functionalized materials decrease with increasing amount of GPTMS, but a desirable pore structure remains when the GPTMS amount increases to 10%. The coexistence of the epoxy groups and the diol groups provides an efficient two-step covalent enzyme immobilization mechanism. The bifunctional PMOs materials exhibit higher papain immobilization efficiency and stability than pure PMOs because of the covalent interaction between the amino groups of papain and the epoxy groups of functionalized PMOs.

Immobilization of enzymes at high load/activity by aqueous electrodeposition of enzyme-tethered chitosan for highly sensitive amperometric biosensing

Electrodeposition has been so widely used to immobilize biomacromolecules, and it is always an important topic to increase the load and activity of the immobilized biomacromolecules. We report here on a new, simple and rather universal method for the highly efficient immobilization of enzymes by aqueous electrodeposition of enzyme-tethered chitosan (CS) for sensitive amperometric biosensing. Glucose oxidase (GOx) is chosen here to examine the proposed protocol in detail. GOx was crosslinked to CS with low-concentration glutaraldehyde (GA, 0.080 wt%), and the electroreduction of added H 2O 2 increased the electrode-surface pH and triggered the electrodeposition of a GOx-GA-CS composite film. The GOx-GA-CS electrodeposition was monitored by an electrochemical quartz crystal microbalance and is theoretically discussed based on an electrogenerated base-to-acid titration model. The prepared first-generation enzyme electrode (CS-GA-GOx/Pt nano/Au) exhibits a current sensitivity as high as 102 μA mM −1 cm −2 at 0.70 V vs SCE, being 13 times that of the CS-GOx/Pt nano/Au prepared similarly but without the GOx-CS precrosslinking. UV–vis spectrophotometric determination of the GOx remains in the supernatant liquids after pH-induced CS precipitation suggested a high enzyme load in the GOx-GA-CS film, and amperometric measurements suggested a negligible decrease in the enzymatic activity of GOx after its reaction with the low-concentration GA. Also, the proposed protocol works well for the precrosslinking manner of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysulfosuccinimide activation, the water-electroreduction-triggered CS electrodeposition, the second-generation biosensing mode, a 5.0-μm-radius Pt ultramicroelectrode, and immobilization of alkaline phosphatase for phenyl phosphate biosensing. The proposed protocol of pretethering the target biomacromolecules to the electrodeposition precusor for immobilization of the biomacromolecule at high load/activity is recommended for wide applications.

Measurement of acetylcholinesterase inhibition using bienzymes immobilized monolith micro-reactor with integrated electrochemical detection

This paper reports a simple μ-FIA based method for the rapid evaluation of acetylcholinesterase inhibition based on bienzymes immobilized monolith micro-reactor, with integrated electrochemical detection. The monolith was prepared inside a micro-fluidic device from two precursors TMOS and MTMOS using a sol–gel method, followed by PEI polymer functionalization and subsequent enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. A bienzyme system containing co-immobilized acetylcholinesterase and choline oxidase was used for the evaluation of enzyme inhibition induced by malaoxon, eserine and methomyl analytes. The proposed method, which gave a LOD of 0.5, 0.2 and 1.0 μM for malaoxon, eserine and methomyl repeatedly, was found to offer several advantages over existing systems including efficient enzyme immobilization, minimal reagent consumption and rapid analysis capability.

Protamine-induced biosilica as efficient enzyme immobilization carrier with high loading and improved stability

β-glucuronidase (GUS) was encapsulated inside mesoporous biosilica prepared via a biomimetic silicification approach. Protamine, a natural cationic polypeptide, was used to induce the condensation of sodium silicate and the enzyme GUS was thus encapsulated during the precipitation process. The enzyme loading efficiency was found to be as high as 100% within its solubility limit in aqueous solution. Furthermore, all the enzyme molecules were tightly entrapped in the biosilica particles without any leaching even under a high ionic strength condition. The biosilica particles displayed a dramatic enrichment effect toward the substrate, baicalin, through nonspecific adsorption. The catalytic activity of the encapsulated GUS was evaluated by converting baicalin into baicalein, two important effective components of herbal medicines. It was found that the thermal and pH tolerance as well as the storage and recycling stability of the encapsulated GUS were significantly enhanced compared with those of the free enzyme. Under the optimum reaction conditions (37 °C, pH 7.0), a high productivity of baicalein of 75% was obtained, only 5% lower than the case for free enzyme counterpart. No appreciable loss in activity was observed after 30-day storage, and 80% of the initial activity could be retained after 4 repeated cycles.

Platinum decorated carbon nanotubes for highly sensitive amperometric glucosesensing

Fine platinum nanoparticles (1–5 nm in diameter) were deposited on functionalizedmulti-walled carbon nanotubes (MWNTs) through a decoration technique. A novel typeof enzymatic Pt/MWNTs paste-based mediated glucose sensor was fabricated.Electrochemical measurements revealed a significantly improved sensitivity (around52.7 µA mM−1 cm−2) for glucose sensing without using any picoampere booster or Faraday cage. In addition,the calibration curve exhibited a good linearity in the range of 1–28 mM of glucoseconcentration. Transmission electron microscopy (TEM) and x-ray photoelectronspectroscopy (XPS) were performed to investigate the nanoscale structure and the chemicalbonding information of the Pt/MWNTs paste-based sensing material, respectively. Theimproved sensitivity of this novel glucose sensor could be ascribed to its higherelectroactive surface area, enhanced electron transfer, efficient enzyme immobilization,unique interaction in nanoscale and a synergistic effect on the current signal from possiblemulti-redox reactions.