Encapsulating Glucose Oxidase Research Articles

Designing Microreactors Resembling Cellular Microenvironment via Polyamine-Mediated Nanoparticle-Assembly for Tuning Glucose Oxidase Kinetics.

Spatial confinement of glucose oxidase (GOx) in the hollow interior of a bioinspired matrix via polyamine mediated silica nanoparticle assembly under environmentally benign conditions is demonstrated herein. In a similarity to the biosilicification processes in diatoms, we use poly(allylamine hydrochloride) (PAH) to direct the assembly of silica nanoparticles on CaCO3 spheres as the removable core. When this assembly process is performed on the CaCO3 spheres, which are preloaded with GOx in a postsynthesis method, microspheres encapsulating GOx are formed. Interestingly, the encapsulated GOx in these microreactors exhibits activity with a Michaelis-Menten constant ( KM) that is 2- to 3-fold less compared with the free enzyme in the solution. While the microenvironment of the organic (PAH)-inorganic (silica) hybrid system can be advantageous for the substrate to interact with enzyme, the effective pH in the vicinity of the entrapped enzyme may also be accountable for the improved activity, resulting in the lower apparent KM and enhanced specificity constant ( kcat/ KM). A 2-fold higher thermal stability of the encapsulated GOx compared with free GOx in solution further demonstrates the efficacy of the integrated architecture. Additionally, the PAH by virtue of its buffering capability allows the microspheres in imparting pH stability to the encapsulated GOx. Therefore, the method is not only a greener process for performing enzyme immobilization but also anticipated to aid in designing microreactors for enhanced enzyme activity.

Encapsulation of glucose oxidase in alginate hollow beads to reduce the fermentable sugars in simulated musts

The treatment of must with glucose oxidase (GOX) is a biotechnological alternative to the production of reduced-alcohol wines. Nevertheless, the low pH of must is a limiting factor for the enzyme's activity. This study reports on a simple immobilization method that uses enzyme encapsulation to overcome that limiting factor. The encapsulation of GOX was acceptably optimized using response surface methodology (RSM). An immobilization yield of 73% was obtained with 22 g L−1 Na-alginate, 20 g L−1 carboxymethylcellulose, and 12 g L−1 CaCl2. The apparent Km value of the immobilized GOX (4.55 mM) was slightly higher than that of the soluble enzyme (3.99 mM) and the activation energy was similar (~ 35 kJ mol−1). The encapsulated enzyme maintained about 68% and 92% of its initial activity at pH 3.0 and 4.0, respectively. Moreover, GOX-loaded alginate hollow beads could be re-used with 7 reaction cycles in model must, with a final reaction efficacy of 37%. GOX was successfully encapsulated in Ca-alginate hollow beads, and their catalytic potential remained unaffected by the immobilization process. In view of these results and the activity of the immobilized enzyme at the acidic pH of the must, this encapsulated biocatalyst is a promising alternative for industrial use in the production of reduced-alcohol wines.

Highly sensitive and stable zwitterionic poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT) glucose biosensor.

A zwitterionic poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT)-based glucose biosensor was fabricated via encapsulating glucose oxidase (GOx) in a one-step electropolymerization method. Integrating conductivity and hydrophilic properties, PSBEDOT provides a great framework for GOx immobilization and stabilization. The anti-fouling, high-sensitivity, and long-term stability properties of PSBEDOT-GOx make it a promising platform for long-term and continuous glucose monitoring.

The study of a fluorescent biosensor based on polyelectrolyte microcapsules with encapsulated glucose oxidase

A fluorescent biosensor is synthesized and described. The biosensor consists of polyelectrolyte microcapsules with glucose oxidase (GOx) entrapped in the cavities and an oxygen-sensitive fluorescent indicator Ru(dpp) immobilized in shells, where Ru(dpp) is tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride. The theoretical activity of the encapsulated GOx and the effect storage time and medium composition have on the stability of sensor microcapsules are determined from polarographic measurements. No change in the activity of the encapsulated enzyme and or its loss to the storage medium are detected over the test period. The dispersion medium (water or a phosphate buffer) are shown to have no effect on the activity of microcapsules with immobilized GOx. The described optical sensor could be used as an alternative to electrochemical sensors for in vitro determination of glucose in the clinically important range of concentrations (up to 10 mmol/L).

Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossing.

In recent years, scientists have created artificial microscopic and nanoscopic self-propelling particles, often referred to as nano- or microswimmers, capable of mimicking biological locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising tasks is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chemical gradients. We report a fully synthetic, organic, nanoscopic system that exhibits attractive chemotaxis driven by enzymatic conversion of glucose. We achieve this by encapsulating glucose oxidase alone or in combination with catalase into nanoscopic and biocompatible asymmetric polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way toward higher-concentration regions. We finally demonstrate that the chemotactic behavior of these nanoswimmers, in combination with LRP-1 (low-density lipoprotein receptor-related protein 1) targeting, enables a fourfold increase in penetration to the brain compared to nonchemotactic systems.

Enzyme-Regulated Fast Self-Healing of a Pillararene-Based Hydrogel.

Self-healing, one of the exciting properties of materials, is frequently used to repair the damage of biological and artificial systems. Here we have used enzymatic catalysis approaches to develop a fast self-healing hydrogel, which has been constructed by dynamic aldimine cross-linking of pillar[5]arene-derivant and dialdehyde-functionalized PEG followed by encapsulation of glucose oxidase (GOx) and catalase (CAT). In specific, the two hydroxyl groups at terminal of PEG4000 are functionalized with benzaldehydes that can interact with amino-containing pillar[5]arene-derivant through dynamic aldimine cross-links, resulting in reversible dynamic hydrogels. Modulus analysis indicated that storage modulus (G') and loss modulus (G″) of the hydrogel increased obviously as the concentration of dialdehyde-functionalized PEG4000 (DF-PEG4000) increased or the pH values decreased. Once glucose oxidase (GOx) and catalase (CAT) are located, the hydrogel could be fast repaired, with self-healing efficiency up to 100%. Notably tensile test showed that the repair process of pillararene-based hydrogel can finish in several minutes upon enzyme catalysis, while it needed more than 24 h to achieve this recovery without enzymes. This enzyme-regulated self-healing hydrogel would hold promise for delivering drugs and for soft tissue regeneration in the future.

Coordinated Membrane Fusion of Proteinosomes by Contact-Induced Hydrogel Self-Healing.

Controlled membrane fusion of proteinosome-based protocells is achieved via a hydrogel-mediated process involving dynamic covalent binding, self-healing, and membrane reconfiguration at the contact interface. The rate of proteinosome fusion is dependent on dynamic Schiff base covalent interchange, and is accelerated in the presence of encapsulated glucose oxidase and glucose, or inhibited with cinnamyl aldehyde due to enzyme-mediated decreases in pH or competitive covalent binding, respectively. The coordinated fusion of the proteinosomes leads to the concomitant transportation and redistribution of entrapped payloads such as DNA and dextran. Silica colloids with amino-functionalized surfaces undergo partial fusion with the proteinosomes via a similar dynamic hydrogel-mediated mechanism. Overall, the strategy provides opportunities for the development of interacting colloidal objects, control of collective behavior in soft matter microcompartmentalized systems, and increased complexity in synthetic protocell communities.

Preparation of Ultrathin, Robust Nanohybrid Capsules through a "Beyond Biomineralization" Method.

Herein, a facile and generic method is developed to prepare ultrathin, robust nanohybrid capsules by manipulating the dynamic structure of supramolecular nanocoatings on CaCO3 sacrificial templates by incorporating a multivalent-anion substitution process into biomineralization. Above the biomineralization level, multivalent anions, for example, phosphate, sulfate, or citrate, are used to initiate the assembly of polyamine into continuous (nonsegregated) polyamine-anion supramolecular nanocoatings on CaCO3 sacrificial templates. When contacting with the sodium silicate solution, the multivalent anions in the supramolecular nanocoatings are substituted by silicate because of the difference in dissociation behavior, facilitating the structure-reconstruction of supramolecular nanocoatings. At the biomineralization level, the substituted silicate can not only bind to the polyamine through electrostatic and hydrogen bonding interactions but also undergo silicification to generate an interpenetrating silica framework. After dissolution of CaCO3, polyamine-silica nanohybrid capsules bearing an ultrathin wall of ∼10-17 nm in thickness are formed, which exhibit a super-high mechanical strength of ∼2337 MPa in elasticity modulus. The capsules are then utilized for bioreactor construction by encapsulating glucose oxidase. The ultrathin capsule wall facilitates the diffusion of substrates/products and elevates the conversion efficiency, whereas the high mechanical strength ensures the structural integrity of the capsules during multiple-cycle reactions. This method can also be applied for the preparation of ultrathin films on planar substrates, which would open a feasible way to prepare nanohybrid materials with different compositions and shapes.

An amperometric glucose oxidase biosensor based on liposome microreactor-chitosan nanocomposite-modified electrode for determination of trace mercury

A biosensor for trace mercury ions based on glucose oxidase (GOD) immobilized on liposome microreactor and chitosan (CS) nanocomposite through layer-by-layer method is described herein. The GOD liposome microreactors (GLMs) were characterized using Fourier transform infrared (FTIR), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and amperometric methods. The results indicated that GLMs were prepared by encapsulating the enzyme GOD in l-α-phosphatidylcholine liposome resulting in spherical bioreactor with a mean diameter of 5.83 ± 0.75 μm. The encapsulation efficiency and drug loading content of the GLMs were about 53.58 ± 0.91 and 41.15 ± 0.95%, respectively. Cyclic voltammogram (CV) was further utilized to explore relevant electrochemical activity on (CS/GLM)8 nanocomposite-modified glassy carbon electrode (GCE). The biosensor based on the (CS/GLM)8-GCE composite films was applied to detect Hg2+ with a broad linear range from 0.5 to 5.00 μmol/L, and the detection limit was brought down to 0.076 μmol/L. The apparent Michaelis-Menten constant, K m, for the enzymatic reaction was 0.37 mmol/L (S/N = 3). Furthermore, the biosensor showed good stability and reproducibility. Such new biosensor based on encapsulation of GOD within liposome microreactors shows great promise for rapid, simple, and cost-effective analysis of Hg2+ in environmental samples.

Introduction to Editorial Board Members: Prof Nicholas A. Peppas

Introduction to Editorial Board Members: Prof Nicholas A. Peppas

Facile Method To Prepare Microcapsules Inspired by Polyphenol Chemistry for Efficient Enzyme Immobilization.

In this study, a method inspired by polyphenol chemistry is developed for the facile preparation of microcapsules under mild conditions. Specifically, the preparation process includes four steps: formation of the sacrificial template, generation of the polyphenol coating on the template surface, cross-linking of the polyphenol coating by cationic polymers, and removal of the template. Tannic acid (TA) is chosen as a representative polyphenol coating precursor for the preparation of microcapsules. The strong interfacial affinity of TA contributes to the formation of polyphenol coating through oxidative oligomerization, while the high reactivity of TA is in charge of reacting/cross-linking with cationic polymer polyethylenimine (PEI) through Schiff base/Michael addition reaction. The chemical/topological structures of the resultant microcapsules are simultaneously characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), etc. The wall thickness of the microcapsules could be tailored from 257±20 nm to 486±46 nm through changing the TA concentration. The microcapsules are then utilized for encapsulating glucose oxidase (GOD), and the immobilized enzyme exhibits desired catalytic activity and enhanced pH and thermal stabilities. Owing to the structural diversity and functional versatility of polyphenols, this study may offer a facile and generic method to prepare microcapsules and other kinds of functional porous materials.

Nano-Encapsulation of Glucose Oxidase Dimer by Graphene

ABSTRACTUsing all-atom molecular dynamics simulations in water environment, it was possible to demonstrate spontaneous and tight encapsulation of glucose oxidase (GOx) dimer by graphene 7 nm x 7 nm sheets linked together by linkers of different width and forming a flower-like or cross-like shapes. The partially overlapping graphene sheets compacted the structure of GOx dimer, bringing the monomers much closer to one another. We found that the most complete wrapping of the enzyme was achieved for the cross-like graphene. Encapsulation can be a useful way to obtain a large contact surface. However, an exceptionally tight binding by the graphene can also influence the positions of amino acids in the enzyme binding site resulting in less efficient catalytic reaction. Furthermore, such extensive encapsulation could block the access of the substrate to the active site of the enzyme. Contrary, a partial encapsulation by graphene using nano-sheets caused only small distortions of GOx structure while the contact surface with graphene was high.

Effect of formulation factors on the bioactivity of glucose oxidase encapsulated chitosan–alginate microspheres: In vitro investigation and mathematical model prediction

Higher reactive oxygen species (ROS) levels in cancer cells than normal cells have long been recognized, which makes cancer cells more susceptible to excess ROS. Thus oxidation (also called pro-oxidant) therapy has been explored as new cancer therapy regimens. To produce additional ROS, e.g. H2O2in situ within tumor, we encapsulated glucose oxidase in chitosan-coated alginate–calcium microspheres (GOX-MS) for locoregional treatment and demonstrated its efficacy against cancer cells in vitro and in vivo. Owing to the complex biological functions of ROS, the production rate and amount of H2O2 are critical to achieve therapeutic benefits without causing normal tissue toxicity. This work was therefore intended to investigate the effect of formulation factors, e.g., particle size and enzyme loading level, on the H2O2 generation kinetics and bioactivity of GOX-MS. In vitro studies revealed that, at the same GOX loading levels, smaller GOX-MS (20μm in diameter) generated more H2O2 and killed more cancer cells than the larger (140μM) GOX-MS. A mathematical model including simultaneous diffusion and enzymatic reaction was developed to describe the release kinetics of generated H2O2 from a GOX-loaded spherical polymeric matrix. Profiles of species concentration, pH, polymer volume fraction and solute diffusivity inside the microspheres were numerically calculated. The model predicted H2O2 release profile was verified by the experimental data. Numerical analysis predicted quantitatively how H2O2 generation increased with increasing GOX loading inside the GOX-MS, glucose concentration in the external solution, or with decreasing particle size. The findings and methodology presented herein are useful for optimizing the design of GOX-MS and applicable to understanding and development of other enzyme-encapsulated polymer microspheres.

A novel amperometric glucose biosensor based on ternary gold nanoparticles/polypyrrole/reduced graphene oxide nanocomposite

A ternary nanocomposite comprised of dispersed small gold nanoparticles (AuNPs) on the surface of reduced graphene oxide (RGO) was achieved via facile wet-chemical routes by using polypyrrole (PPy) as a linker. Highly dispersed small AuNPs supported on PPy/RGO surface have been evidenced by the TEM and SEM characterization. The composite exhibited excellently electrocatalytic property toward O2 reduction compared with RGO/PPy, RGO and PPy. Based on the oxygen reduction activity and compatibility of the composite, its prospective in biosensing was further exemplified. Upon encapsulation of glucose oxidase via chitosan cross linking, a glucose electrochemical biosensor was constructed. The biosensor manifested a reproducible amperometric response to glucose in a linear range of 0.2–1.2mM (R=0.986) with a sensitivity of 123.8μAmM−1cm−2. The results indicate that the ternary nanocomposite is promising in biosensor fabrication.

Rate of solute incorporation to liposomes evaluated from encapsulated enzymes activities.

There are numerous studies on systems comprising an enzyme encapsulated in unilamellar liposomes and its substrate initially present in the external aqueous media. Most of these studies are focused on enzyme stability and activity in a restricted media. However, the rate of the process is also determined by the capacity of the substrate to permeate towards the liposome inner pool. In spite of this, there are few studies aimed at a quantitative evaluation of the substrate permeation rate and its lifetime inside the liposome pool. In the present work, we describe, in terms of a very simple mechanism, the permeation of glucose and hydrogen peroxide in DPPC unilamellar liposomes. To this aim, we evaluated the rate of the process employing encapsulated glucose oxidase and catalase in the kinetic diffusion controlled limit. Under this condition, the rate of the process becomes zero order in the enzyme and allows a direct evaluation of the rate constant for the permeation process and the lifetime of a substrate molecule incorporated into the liposome inner pool.

Enzyme-modified carbon-fiber microelectrode for the quantification of dynamic fluctuations of nonelectroactive analytes using fast-scan cyclic voltammetry.

Neurotransmission occurs on a millisecond time scale, but conventional methods for monitoring nonelectroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, nonelectroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically generated H2O2 without the need for additional strategies to eliminate interfering agents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM(-1) and 13.1 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many nonelectroactive enzyme substrates.

Volume Change Behavior of Poly(l-lysine-alt-terephthalic acid) Microcapsules Encapsulating Glucose Oxidase

Abstract Glucose-sensitive microcapsules, whose volume changed in response to glucose, were successfully prepared by encapsulating glucose oxidase (GOx) into poly(l-lysine-alt-terephthalic acid) (PPL) microcapsules. The GOx–PPL microcapsules decreased in size in a glucose solution, after a certain lag time. The shrinking rate of the capsules showed a linear correlation with glucose concentration, while the lag time showed a logarithmical correlation with the glucose concentration.

Nafion coating the ferrocenylalkanethiol and encapsulated glucose oxidase electrode for amperometric glucose detection

This paper describes the fabrication and application of a complex electrode--Nafion film coating ferrocenylalkanethiol (FcC(11)SH) and encapsulated glucose oxidase (GOD) on a gold electrode. FcC(11)SH is employed as a mediator enabling the electron transfer between GOD and the electrode, GOD is encapsulated in polyacrylamide gel to improve the stability of the enzyme, and the Nafion film is coated on the modified electrode to eliminate interferents such as ascorbic acid, uric acid and acetaminophen in amperometric glucose detection. It is noticed that such a complex electrode exhibits excellent catalytic activity for glucose oxidation, and preserves the native structure of GOD and therefore its enzymatic activity. The encapsulated GOD retains more than 80% of its original biocatalytic activity even after 24 days, much longer than that of naked GOD molecules attached directly to the electrode. The oxidation peak current at the modified electrode shows a linear relationship with the glucose concentration in the range from 0.05 to 20 mM with a detection limit of 2.4 μM. In addition, the electrode displays a rapid response and good reproducibility for glucose detection, and has been successfully employed for glucose detection in blood plasma samples.

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au shell@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab 2) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au shell@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au shell@GOD were performed by one-pot assembly of Fc@Ab 2 and GOD on the surface of Au shell@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL −1 with the detection limit down to 6.7 pg mL −1. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.

Role of porosity in tuning the response range of microsphere-based glucose sensors

Luminescent microspheres encapsulating glucose oxidase have recently been developed as implantable glucose sensors. Previous work has shown that the response range and sensitivity can be tuned by varying the thickness and composition of transport-controlling nanofilm coatings. Nevertheless, the linear response range of these sensors falls significantly below the desired clinical range for in vivo monitoring. We report here an alternative means of tuning the response range by adjusting microsphere porosity. A reaction-diffusion model was first used to evaluate whether increased porosity would be expected to extend the response range by decreasing the flux of glucose relative to oxygen. Sensors exhibiting linear response ( R 2 > 0.90) up to 600 mg/dL were then experimentally demonstrated by using amine-functionalized mesoporous silica microspheres and polyelectrolyte nanofilm coatings. The model was then used for sensor design, which led to the prediction that sensors constructed from ∼12 μm microspheres having an effective porosity between 0.005 and 0.01 and ∼65 nm transport-limiting coatings would respond over the entire physiological glucose range (up to 600 mg/dL) with maximized sensitivity.