Direct electron transfer of glucose oxidase promoted by carbon nanotubes is without value in certain mediator-free applications

We have investigated the direct electron transfer (DET) promoted by carbon nanotubes (CNTs) on an electrode containing immobilized glucose oxidase (GOx) with the aim to develop a third-generation glucose biosensor and a mediator-free glucose biofuel cell anode. GOx was immobilized via chitosan (CS) on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Cyclic voltammetric revealed that the GOx on the surface of such an electrode is unable to simultaneously demonstrate DET with the electrode and to retain its catalytic activity towards glucose, although the MWCNTs alone can promote electron transfer between GOx and electrode. This is interpreted in terms of two types of GOx on the surface, the distribution and properties of which are quite different. The first type exhibits DET capability that results from the collaboration of MWCNTs and metal impurities, but is unable to catalyze the oxidation of glucose. The second type maintains its glucose-specific catalytic capability in the presence of a mediator, which can be enhanced by MWCNTs, but cannot undergo DET with the electrode. As a result, the MWCNTs are capable of promoting the electron transfer, but this is without value in some mediator-free applications such as in third-generation glucose biosensors and in mediator-free anodes for glucose biofuel cells.

Direct electrodeposition of a biocomposite consisting of reduced graphene oxide, chitosan and glucose oxidase on a glassy carbon electrode for direct sensing of glucose

We have electrodeposited a composite film consisting of graphene oxide, chitosan and glucose oxidase directly on a glassy carbon electrode (GCE) through electrochemical reduction of a solution of the 3 components under controlled direct electrical potential. The procedure takes only several minutes, and the thickness of the resulting film is uniform and controllable. The GOx has uncompromised bioactivity and exhibits reversible 2-proton and 2-electron transfer in presence of glucose. It therefore can be used amperometric sensing of glucose. The biosensor has a fast response (<3 s), a detection limit of 0.4 μM (which is 50-fold lower compared to the biosensor prepared by drop-casting solutions of the same materials onto an GCE), and a linear response in the 0.4 μM to 2 mM concentration range (which again is much better than that of the biosensor prepared by the drop-casting method). Other features include high reproducibility, long-time storage stability, and satisfactory selectivity. We presume that the direct single-step electrodeposition of this nanocomposite offers a promising approach towards novel types of highly sensitive and stable electrochemical biosensors.

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

An enzymatic glucose biosensor based on a glassy carbon electrode modified with manganese dioxide nanowires

A glassy carbon electrode was modified with β-manganese dioxide (β-MnO₂), and glucose oxidase (GOx) was immobilized on its surface. The β-MnO₂ nanowires were prepared by a hydrothermal method and characterized by scanning electron microscopy and powder X-ray diffraction. They were then dispersed in Nafion solution and cast on the glassy carbon electrode (GCE) to form an electrode modified with β-MnO₂ nanowires that exhibits improved sensitivity toward hydrogen peroxide. If GOx is immobilized in the surface, the β-MnO₂ acts as a mediator, and Nafion as a polymer backbone. The fabrication process was characterized by electrochemical impedance spectroscopy, and the sensor and its materials were characterized by cyclic voltammetry and amperometry. The biosensor enables amperometric detection of glucose with a sensitivity of 38.2 μA?·?mM^?1?·?cm^?2, and a response time of?<?5 s. This study also demonstrates the feasibility of realizing inexpensive, reliable, and high-performance biosensors using MnO₂ nanowires.

Simple construction of an enzymatic glucose biosensor based on a nanocomposite film prepared in one step from iron oxide, gold nanoparticles, and chitosan

The one-step synthesis is reported of a nanofilm composed of iron oxide and gold nanoparticles in a chitosan matrix that can act as a novel matrix for the immobilization of glucose oxidase (GOx) to fabricate a glucose biosensor. The use for the composite film strongly increased the effective electrode surface for loading of GOx. The size and shape of the iron oxide nanoparticles were examined by transmission electron micrograph. Direct electron transfer and electrocatalysis by GOx was investigated via cyclic voltammetry and chronoamperometry. Under optimized conditions, the biosensor has a response time of 6?s and a linear response in the range between 3???M and 0.57?mM of glucose, with a detection limit of 1.2???M at a signal-to-noise ratio of 3. This novel and disposable mediatorless glucose biosensor may form the basis for a future mass-produced glucose biosensor.

Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM^?1 cm^?2) and good linearity in the range from 0.05 to 15 mM (r?=?0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer.

Tin disulfide nanoflakes decorated with gold nanoparticles for direct electrochemistry of glucose oxidase and glucose biosensing

Previously, we have prepared nanoflake-like tin disulfide (SnS2) and used for the immobilization of proteins and biosensing. We have now modified an electrode with a composite consisting of nanoflake-like SnS2 decorated with gold nanoparticles (Au-NPs) and have immobilized glucose oxidase (GOx) on its surface in order to study its direct electrochemistry. Scanning electron microscopy, electrochemical impedance spectroscopy, Fourier transform IR spectroscopy and cyclic voltammetry were used to examine the interaction between GOx and the AuNP-SnS2 film. It is shown that the composite film has a larger surface area and offers a microenvironment that facilitates the direct electron transfer between enzyme and electrode surface. The immobilized enzyme retains its bioactivity and undergoes a surface-controlled, reversible 2-proton and 2-electron transfer reaction, with an apparent electron transfer rate constant of 3.87 s ^-1. Compared to the nanoflake-like SnS2-based glucose sensor, the GOx-based biosensor exhibits a lower detection limit (1.0 :M), a better sensitivity (21.8 mA?M ^-1 ?cm ^-2), and a wider linear range (from 0.02 to 1.3 mM). The sensor displays excellent selectivity, good reproducibility, and acceptable stability. It was successfully applied to reagentless sensing of glucose at ?0.43 V.

Reagentless amperometric glucose biosensor based on the immobilization of glucose oxidase on a ferrocene@NaY zeolite composite

Ferrocene (Fc) was encapsulated in the cavities of a NaY zeolite by vapor diffusion via sublimation at below 100?°C. The resulting Fc@NaY zeolite composite was investigated by power X-ray diffraction, diffuse reflectance UV?Cvis and FT-IR spectroscopy, and by cyclic voltammetry. The results indicated that Fc was encapsulated into the zeolite whose microporous structure had remained intact. The Fc in the silica matrix had retained its electroactivity and did not leach out. A glucose biosensor was obtained by immobilization of the modified zeolite and glucose oxidase on a carbon paste electrode. It displays a linear response to glucose (from 0.8???M to 4.0?mM), a detection limit of 0.2???M, and a response time of 4?s. The good performance of the biosensor is ascribed to the biocompatibility of the zeolite and presence of Fc which facilitates the electron transfer from the enzyme to the surface of the electrode.

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Glucose biosensor based on glucose oxidase immobilized on unhybridized titanium dioxide nanotube arrays

A glucose biosensor has been fabricated by immobilizing glucose oxidase (GOx) on unhybridized titanium dioxide nanotube arrays using an optimized cross-linking technique. The TiO₂ nanotube arrays were synthesized directly on a titanium substrate by anodic oxidation. The structure and morphology of electrode material were characterized by X-ray diffraction and scanning electron microscopy. The electrochemical performances of the glucose biosensor were conducted by cyclic voltammetry and chronoamperometry measurements. It gives a linear response to glucose in the 0.05 to 0.65 mM concentration range, with a correlation coefficient of 0.9981, a sensitivity of 199.6 μA mM^?1 cm^?2, and a detection limit as low as 3.8 µM. This glucose biosensor exhibited high selectivity for glucose determination in the presence of ascorbic acid, sucrose and other common interfering substances. This glucose biosensor also performed good reproducibility and long-time storage stability. This optimized cross-linking technique could open a new avenue for other enzyme biosensors fabrication.

Amperometric glucose biosensor based on electrodeposition of platinum nanoparticles onto covalently immobilized carbon nanotube electrode

A new amperometric biosensor for glucose was developed based on adsorption of glucose oxidase (GOx) at the gold and platinum nanoparticles-modified carbon nanotube (CNT) electrode. CNTs were covalently immobilized on gold electrode via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM). The fabricated GOx/Au_nano/Pt_nano/CNT electrode was covered with a thin layer of Nafion to avoid the loss of GOx in determination and to improve the anti-interferent ability. The immobilization of CNTs on the gold electrode was characterized by quartz crystal microbalance technique. The morphologies of the CNT/gold and Pt_nano/CNT/gold electrodes have been investigated by scanning electron microscopy (SEM), and the electrochemical performance of the gold, CNT/gold, Pt_nano/gold and Pt_nano/CNT/gold electrodes has also been studied by amperometric method. In addition, effects of electrodeposition time of Pt nanoparticles, pH value, applied potential and electroactive interferents on the amperometric response of the sensor were discussed.

The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for glucose in the absence of a mediator. The linear range was from 0.5 to 17.5 mM with correction coefficient of 0.996. The biosensor had good reproducibility and stability for the determination of glucose.     

Reagentless d-sorbitol biosensor based on d-sorbitol dehydrogenase immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite

A reagentless d-sorbitol biosensor based on NAD-dependent d-sorbitol dehydrogenase (DSDH) immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD⁺ cofactor with DSDH in a sol–gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of d-sorbitol at 0.2 V with a sensitivity of 8.7?μA?mmol^?1?L?cm^?2 and a detection limit of 0.11 mmol?L^?1. Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

Biosensor for aluminium(III) based on its inhibition of α-chymotrypsin immobilized on a screen-printed carbon electrode modified with gold nanoparticles

We report on an amperometric assay for Al(III) ions that is based on the inhibition of the enzyme α-chymotrypsin. Screen-printed carbon electrodes modified with gold nanoparticles were used as solid supports for the immobilization of the enzyme. The amperometric response of the synthetic enzyme substrate substrate N-benzoyl-L-tyrosine ethyl ester is affected by Al(III) ions, and this leads to a decrease in the amperometric oxidation current. The assay has a detection limit of 3.3?μM of Al(III). The repeatability and reproducibility of the method are 6.9% (n?=?3) and 6.4% (n?=?5), respectively. Main interferents include Mo(VI), W(VI) and Fe(III) ions. The method was successfully applied to the determination of Al(III) in tap water.

Electrochemical detection of in situ DNA damage induced by enzyme-catalyzed Fenton reaction. Part I: in phosphate buffer solution

We report on a biosensor for the electrochemical detection of the damage of DNA and of antioxidant protecting DNA. The biosensor was constructed by co-immobilization of DNA and glucose oxidase (GOx) on a glassy carbon electrode. Under aerobic conditions, GOx catalyzes the oxidation of glucose, and the hydrogen peroxide produced reacts with ferrous ions in a Fenton-type reaction to generate hydroxy radical. This was validated by UV–vis spectroscopy. The hydroxy radical can cause serious oxidative damage to DNA, and this can be detected by square wave voltammetry of the electroactive indicator Co(bpy) ₃ ³⁺ . The effects of pH value, incubation time, and the concentration of glucose and ferrous ion were optimized. The effects of the antioxidants ascorbic acid and aloe emodin on DNA damage were also investigated within the concentration range from 0.05 to 200?μM. This work provides an in-vitro model system to mimic the processes in oxidative DNA damage by a simple electrochemical approach.

Novel glucose biosensor based on a glassy carbon electrode modified with hollow gold nanoparticles and glucose oxidase

A novel glucose biosensor is presented as that based on a glassy carbon electrode modified with hollow gold nanoparticles (HGNs) and glucose oxidase. The sensor exhibits a better differential pulse voltammetric response towards glucose than the one based on conventional gold nanoparticles of the same size. This is attributed to the good biological conductivity and biocompatibility of HGNs. Under the optimal conditions, the sensor displays a linear range from 2.0?×?10^?6 to 4.6?×?10^?5?M of glucose, with a detection limit of 1.6?×?10^?6?M (S/N?=?3). Good reproducibility, stability and no interference make this biosensor applicable to the determination of glucose in samples such as sports drinks.

Electrochemiluminescent detection of the hybridization of oligonucleotides using an electrode modified with nanocomposite of carbon nanotubes and gold nanoparticles

We report on an electrochemiluminescent (ECL) sensing technique for the detection of the hybridization between oligonucleotides. A glassy carbon electrode was first functionalized with a composite prepared from gold nanoparticles and carbon nanotubes, and a sensor was then constructed by immobilizing the probing oligonucleotide. The ECL of luminol acts as the sensing signal. It is quenched, to a different degree, by the hybridized double strands of the oligonucleotide depending on the match status. The slope of the ECL response as a function of the status of hybridization drops with increasingly matched hybridization. The response is attributed to the interaction between luminol and the strands of oligomers, and also related to the reduction of reactive oxygen species.

Optical determination of glucose and hydrogen peroxide using a nanocomposite prepared from glucose oxidase and magnetite nanoparticles immobilized on graphene oxide

Fe₃O₄ nanoparticles were deposited on sheets of graphene oxide (GO) by a precipitation method, and glucose oxidase (GOx) was then immobilized on this material to produce a GOx/Fe₃O₄/GO magnetic nanocomposite containing crosslinked enzyme clusters. The 3-component composite functions as a binary enzyme that was employed in a photometric method for the determination of glucose and hydrogen peroxide where the GOx/Fe₃O₄/GO nanoparticles cause the generation of H₂O₂ which, in turn, oxidize the substrate N,N-diethyl-p-phenylenediamine to form a purple product with an absorption maximum at 550 nm. The absorbance at 550 nm can be correlated to the concentration of glucose and/or hydrogen peroxide. Under optimized conditions, the calibration plot is linear in the 0.5 to 600 μM glucose concentration range, and the detection limit is 0.2 μM. The respective plot for H₂O₂ ranges from 0.1 to 10 μM, and the detection limit is 0.04 μM. The method was successfully applied to the determination of glucose in human serum samples. The GOx/Fe₃O₄/GO nanoparticles are reusable.

Direct electrochemistry of glucose oxidase and sensing glucose using a screen-printed carbon electrode modified with graphite nanosheets and zinc oxide nanoparticles

We have studied the direct electrochemistry of glucose oxidase (GOx) immobilized on electrochemically fabricated graphite nanosheets (GNs) and zinc oxide nanoparticles (ZnO) that were deposited on a screen printed carbon electrode (SPCE). The GNs/ZnO composite was characterized by using scanning electron microscopy and elemental analysis. The GOx immobilized on the modified electrode shows a well-defined redox couple at a formal potential of ?0.4 V. The enhanced direct electrochemistry of GOx (compared to electrodes without ZnO or without GNs) indicates a fast electron transfer at this kind of electrode, with a heterogeneous electron transfer rate constant (K_s) of 3.75 s^?1. The fast electron transfer is attributed to the high conductivity and large edge plane defects of GNs and good conductivity of ZnO-NPs. The modified electrode displays a linear response to glucose in concentrations from 0.3 to 4.5 mM, and the sensitivity is 30.07 μA mM^?1 cm^?2. The sensor exhibits a high selectivity, good repeatability and reproducibility, and long term stability. Figure

Graphical representation for the fabrication of GNs/ZnO composite modified SPCE and the immobilization of GOx     

A hydrogen peroxide biosensor based on direct electron transfer from hemoglobin to an electrode modified with Nafion and activated nanocarbon

A biosensor for hydrogen peroxide (HP) was developed by immobilizing hemoglobin on a glassy carbon electrode modified with activated carbon nanoparticles/Nafion. The characteristics of the sensor were studied by UV?Cvis spectroscopy and electrochemical methods. The immobilized Hb retained its native secondary structure, undergoes direct electron transfer (with a heterogeneous rate constant of 3.37?±?0.5?s^?1), and displays excellent bioelectrocatalytic activity to the reduction of HP. Under the optimal conditions, its amperometric response varies linearly with the concentration of HP in the range from 0.9???M to 17???M. The detection limit is 0.4???M (at S/N?=?3). Due to the commercial availability and low cost of activated carbon nanoparticles, it can be considered as a useful supporting material for construction of other third-generation biosensors.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.