Covalent Attachment Of Glucose Oxidase Research Articles

Plasma‐functionalized Highly Aligned CNT‐based Biosensor for Point of Care Determination of Glucose in Human Blood Plasma

AbstractIn this study, a new method for modification of vertically aligned carbon nanotube arrays (VACNTs) for selective detection of glucose was developed. VACNTs were grown by chemical vapor deposition method on a silicon substrate deposited with alumina as a buffer layer and iron as a catalyst using radio frequency (RF) sputtering and electron beam evaporation, respectively. The surface of the electrode was modified with electrodeposition of polyaniline (PANI) followed by covalent attachment of glucose oxidase (GOx). The electrode was characterized using field emission scanning electron microscopy (FESEM), micro‐Raman spectroscopy, and attenuated total reflectance Fourier transform infrared spectrometer (ATR‐FTIR) techniques. Cyclic voltammetry and differential pulse voltammetry were used to investigate the electrochemical behavior of the electrode. The fabricated electrode was successfully employed as a point‐of‐care (POC) biosensor for the detection of glucose in human blood plasma. The detection limit was 1.1 μM, and the sensitivity was 620 μA mM−1 cm−2 at the linear range of 2–426 μM.

Oxidoreductase Immobilization on Magnetic Nanoparticles

The aim of this work is to develop the synthesis of the magnetically separable biocatalyst for enzymatic oxidation of D-glucose to D-gluconic acid with high product yields. The biocatalyst support is based on magnetite particles (MPs) synthesized by coprecipitation and coated with the amino- silica layer to facilitate further functionalization. This functionalization involves the attachment of the glutaric dialdehyde linker followed by the covalent attachment of glucose oxidase (GOx, an enzyme of oxidoreductase group) via its amino groups. TEM, XRD, and magnetic measurements were performed for initial MPs and GOx biocatalyst. The biocatalyst activity was studied in the oxidation reaction of D-glucose to D-gluconic acid. The biocatalyst synthesis proposed in this work allowed to create a highly effective system. It was found that GOx immobilized on the modified MPs was the most active biocatalytic system in comparison with other inorganic support and showing 88% of gluconic acid yield.

Insights into Sustainable Glucose Oxidation Using Magnetically Recoverable Biocatalysts

Here, we developed magnetically recoverable biocatalysts for enzymatic oxidation of d-glucose to d-gluconic acid with high product yields. The catalyst support is based on nanoparticle clusters (NPCs) composed of magnetite particles and coated with the amino terminated silica layer to facilitate further functionalization. It involves the attachment of the glutaraldehyde linker followed by the covalent attachment of glucose oxidase (GOx) via its amino groups. It was established that the NPCs with a diameter of ∼430 nm attach 33% more GOx molecules than NPCs with a diameter of ∼285 nm, although the surface area of the former is lower than that of the latter. At the same time, the biocatalyst based on the smaller NPCs shows higher relative activity of 94% than that (87%) of the biocatalyst based on the larger NPCs, both at 50 °C and pH 7 (optimal reaction conditions). This surprising result has been explained by a combination of two major factors such as GOx crowding on the support surface which should preve...

A glucose biosensor based on direct electron transfer of glucose oxidase immobilized onto glassy carbon electrode modified with nitrophenyl diazonium salt

This study reports a novel, simple and fast approach for construction of a highly stable glucose biosensor based on the immobilization of glucose oxidase (GOx) onto a glassy carbon electrode (GCE) electrografted with 4-aminophenyl (AP) by diazonium chemistry. Aminophenyl was used as cross-linker for covalent attachment of glucose oxidase to the electrode surface. Cyclic voltammograms of the GOx-modified GCE in phosphate buffer solution exhibited a pair of well-defined redox peaks, attesting the direct electron transfer (DET) of GOx with the underlying electrode. The proposed biosensor could be used to detect glucose based on the consumption of O2 with the oxidation of glucose catalyzed by GOx and exhibited a wide linear range of glucose from 0.05mM to 4.5mM and low detection limit of 10μM. The surface coverage of active GOx, heterogeneous electron transfer rate constant (ks) and Michaelis–Menten constant (KM) of immobilized GOx were 1.23×10−12molcm−2, 4.25s−1 and 2.95mM, respectively. The great stability of this biosensor, technically simple and possibility of preparation at short period of time make this method suitable for fabrication of low-cost glucose biosensors.

Glucose Biosensor Based on Immobilization of Glucose Oxidase on Silica Nanoparticles Modified Au Electrode

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a silica nanoparticle monolayer modified gold electrode. Electrochemical impedance spectroscopy (EIS) of ferrocyanide followed the assembly process and verified the successful immobilization of GOx on silica nanoparticle modified on gold electrodes. Cyclic voltammetry (CV), performed in the presence of excess glucose and artificial redox mediator (ferrocenemethanol), allowed to quantify the surface concentration of electrically wired enzyme. The signal of proposed electrode was more than 2.5 times of that on electrode lacking silica nanoparticles. As a result, silica nanoparticles are a good biocompatible solid support for enzyme immobilization.

Construction of a Novel Glucose Biosensor Based on Covalent Immobilization of Glucose Oxidase on Poly(glycidyl methacrylate‐co‐vinylferrocene)

AbstractA novel glucose biosensor was constructed via direct covalent attachment of glucose oxidase onto epoxy group containing polymeric electron transfer mediator, Poly(glycidyl methacrylate‐co‐vinylferrocene). A copolymer of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios has been prepared by free radical copolymerization. These copolymers have been utilized as polymeric mediators for amperometric glucose sensing. The catalytic electrochemistry of the enzyme electrode with the copolymer was investigated. Copolymer acts as an electron transfer mediator between the redox center of Glucose oxidase (GOx) and the electrode. The stability, reusability, pH and temperature response of the biosensor as well as its kinetic parameter have also been studied.

Fabrication of an Implantable Fine Needle-Type Glucose Sensor Using γ-Polyglutamic Acid

Implantable fine needle-type glucose sensors with an outer diameter of less that 0.2 mm were fabricated using a low-cost and non-animal origin polyamide, gamma-polyglutamic acid (PGA) as a glucose oxidase (GOx) immobilizing material. Two types of PGA, gamma-polyglutamic acid (PGAH) and gamma-polyglutamic acid sodium salt (PGANa), were employed to prepare GOx immobilized film by the covalent attachment of GOx using water-soluble carbodiimide (EDC). Nafion/cellulose acetate composite film and polyurethane/polydimethylsiloxane composite film were employed as a permselective inner film and a biocompatible outer film, respectively. The procedure of enzyme-immobilized film fabrication affected the stability of the sensor; that is, GOx immobilized film prepared by pouring a mixture solution of GOx and EDC on a PGA precoated surface showed higher sensor stability than that prepared by pouring a mixture solution of GOx, PGA and EDC. Although, obvious differences in the sensor properties were not observed between the use of PGANa and PGAH, the electrode prepared with PGAH had a lower swelling degree. The glucose sensors prepared with both PGANa and PGAH were practically not affected by the existence of electroactive compounds, such as uric acid, and provided long-term stability for approximately 5 weeks. These sensors also showed good performance in horse serum.

Impedance study of the hybrid molecule alginate–pyrrole: Demonstration as host matrix for the construction of a highly sensitive amperometric glucose biosensor

The aim of this paper is to report the characterization and application of a new and attractive hybrid molecule (alginate modified with pyrrole) alginate-pyrrole (Alg–py) with glucose oxidase (GOx) to create a highly responsive glucose biosensor. The biosensor was built by covalent attachment of GOx with the Alg-py matrix, followed by pyrrole polymerization, then casting the biocomposite onto the surface of a platinum electrode (Pt). The unique three-dimensional structure, porous morphology, conductive setup, mechanical firmness and biocompatibility resulted in low response time (4 s), high sensitivity value (16 μA mM −1 cm −2) and a very low enzyme load (9.2 U).

Improved sensitivity and stability of amperometric enzyme microbiosensors by covalent attachment to gold electrodes

Covalent attachment of glucose oxidase to a pre-activated 16-mercaptohexadecanoic acid at a gold ultramicroelectrode surface improves sensitivity, stability, and reproducibility of enzyme-based amperometric microbiosensors. Self-assembled monolayers of the N-hydroxysuccinimide ester of 16-mercaptohexandecanoic acid (NHS-MHA) at gold electrodes enable spontaneous covalent linking of glucose oxidase to the gold surface of ultramicroelectrodes. By self-assembling NHS-MHA for 30min, approximately 93% of the electrode surface is covered, thereby maximizing both the number of attachment sites for glucose oxidase, and sufficient diffusion of hydrogen peroxide to the gold electrode. The glucose oxidase reaction with NHS-MHA was optimized at pH of 6.5, and at a temperature of 43°C, resulting in a surface concentration of 6.8±0.6×1011 enzymemoleculescm−2. Thus obtained amperometric microbiosensors were calibrated in the range of 1–10mM providing excellent correlation with the theoretical prediction of the microbiosensor response. The reported sensitivity of these microbiosensors documents an improvement by one order of magnitude compared to other approaches for covalent enzyme attachment. This is attributed to the NHS-MHA layer spacing the enzymatic recognition interface further from the electrode surface, thereby minimizing quenching of the enzyme activity.

Critical assessment of the Quartz Crystal Microbalance with Dissipation as an analytical tool for biosensor development and fundamental studies: Metallophthalocyanine–glucose oxidase biocomposite sensors

One of the challenges in electrochemical biosensor design is gaining a fundamental knowledge of the processes underlying immobilisation of the molecules onto the electrode surface. This is of particular importance in biocomposite sensors where concerns have arisen as to the nature of the interaction between the biological and synthetic molecules immobilised. We examined the use of the Quartz Crystal Microbalance with Dissipation (QCM-D) as a tool for fundamental analyses of a model sensor constructed by the immobilisation of cobalt(II) phthalocyanine (TCACoPc) and glucose oxidase (GOx) onto a gold-quartz electrode (electrode surface) for the enhanced detection of glucose. The model sensor was constructed in aqueous phase and covalently linked the gold surface to the TCACoPc, and the TCACoPc to the GOx, using the QCM-D. The aqueous metallophthalocyanine (MPc) formed a multi-layer over the surface of the electrode, which could be removed to leave a monolayer with a mass loading that compared favourably to the theoretical value expected. Analysis of frequency and dissipation plots indicated covalent attachment of glucose oxidase onto the metallophthalocyanine layer. The amount of GOx bound using the model system compared favourably to calculations derived from the maximal amperometric functioning of the electrochemical sensor (examined in previously-published literature, Mashazi, P.N., Ozoemena, K.I., Nyokong, T., 2006. Electrochim. Acta 52, 177–186), but not to theoretical values derived from dimensions of GOx as established by crystallography. The strength of the binding of the GOx film with the TCACoPc layer was tested by using 2% SDS as a denaturant/surfactant, and the GOx film was not found to be significantly affected by exposure to this. This paper thus showed that QCM-D can be used in order to model essential processes and interactions that dictate the functional parameters of a biosensor.

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme ( Γ E 0) on the basis of kinetic models reported in literature. The Γ E 0 on proposed electrode was high to 4.1×10 −12 mol·cm −2, which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0×10 −5–5.7×10 −3 M with a sensitivity of 8.8 μA·mM −1·cm −2 and a detection limit of 8.2 μM. The apparent Michaelis–Menten constant ( K m app) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.

The Influence of the Underlying Gold Substrate on Glucose Oxidase Electrodes Fabricated Using Self-Assembled Monolayers

The influence of the underlying gold surface topography on the response of enzyme electrodes fabricated by covalent attachment of glucose oxidase to a 3-mercaptopropionic acid self-assembled monolayer was assessed. Six different gold substrates were prepared. The bare substrates were evaluated for roughness and defects using atomic force microscopy (AFM) and scanning tunneling microscopy (STM). Despite large variations in surface roughness, the shapes of the enzyme electrode calibration plots indicate that approximately the same amount of MPA was adsorbed on the surface and from electrochemical stripping experiments the same number of active enzyme molecules were attached. However, compared with the smoother surfaces, enzyme electrodes fabricated on surfaces with high microscopic roughness had a smaller linear range but a similar current sensitivity in the linear portion of the calibration plot. Tapping-mode AFM images of the enzyme modified surfaces showed that the enzyme adsorbed onto the surface as clusters of five or six enzyme molecules. The formation of these clusters did not appear to be influenced by the surface roughness.

Reticulated vitreous carbon electrode materials chemically modified with immobilized enzyme

The direct covalent attachment of glucose oxidase to the surface of reticulated vitreous carbon (RVC), a honeycomb carbonaceous material, is reported. The activity of the bound enzyme on different types of RVC, and the activity of adsorbed blanks are presented.