Direct electrochemistry of glucose oxidase assembled on graphene and application to glucose detection

The direct electrochemistry of glucose oxidase (GOx) integrated with graphene was investigated. The voltammetric results indicated that GOx assembled on graphene retained its native structure and bioactivity, exhibited a surface-confined process, and underwent effective direct electron transfer (DET) reaction with an apparent rate constant (k_s) of 2.68 s⁻¹. This work also developed a novel approach for glucose detection based on the electrocatalytic reduction of oxygen at the GOx-graphene/GC electrode. The assembled GOx could electrocatalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction current decreased, which could be used for glucose detection with a high sensitivity (ca. 110 ± 3 μA mM⁻¹ cm⁻²), a wide linear range (0.1-10 mM), and a low detection limit (10 ± 2 μM). The developed approach can efficiently exclude the interference of commonly coexisting electroactive species due to the use of a low detection potential (−470 mV, versus SCE). Therefore, this study has not only successfully achieved DET reaction of GOx assembled on graphene, but also established a novel approach for glucose detection and provided a general route for fabricating graphene-based biosensing platform via assembling enzymes/proteins on graphene surface.     

Continuous glucose monitoring using enzyme-immobilized platinized diamond microfiber electrodes

A sensitive and stable glucose biosensor for in vivo monitoring has been developed using boron-doped diamond microfiber (BDDMF) electrodes. The electrodes were modified with platinum nano-particles to detect H₂O₂, which was enzymatically produced by glucose oxidase (GOx) immobilized on the electrode surface. The platinum-modified BDDMF (Pt-BDDMF) electrodes exhibited much higher sensitivity compared to Pt-microfiber electrodes, Pt electrodes and Pt-modified diamond thin film electrodes. Deposition conditions for Pt nano-particles on the BDDMF electrodes and immobilization of GOx were optimized. GOx/overoxidized polypyrrole (OPPy)/Pt-modified BDDMF electrodes were applied for continuous interference-free glucose monitoring. Amperometric measurements of glucose showed a linear response in the range of 1-70 mM, with an R.S.D. of 3.7% for five injections of 100 μM glucose. The electrodes exhibited good stability over 3 months with no detected anodic current for ascorbic acid (AA), which is an interfering compound.     

Neodymium (III) hexacyanoferrate (II) nanoparticles induced by enzymatic reaction and their use in biosensing of glucose

The formation of neodymium (III) hexacyanoferrate (II) (NdHCF) nanoparticles (NPs) on the surface of carbon-paste electrode induced by enzymatic reaction was described and characterized. The conditions for biosensing of glucose were optimized through various experiments. Results showed that the optimized condition of the glucose oxidase (GOx)-induced NdHCF NPs for the biosensing of glucose were 2.0 mM Nd³⁺, 40.0 mM Fe(CN)₆³⁻ and 20 μg/mL GOx. The biocatalyzed generation of NdHCF NPs in the presence of O₂/glucose and GOx enabled the development of an electrochemical biosensor for glucose. Furthermore, this system avoids the interferences from other species for the biosensing of glucose.     

Novel amperometric glucose biosensor based on an ether-linked cobalt(II) phthalocyanine-cobalt(II) tetraphenylporphyrin pentamer as a redox mediator

The development of cobalt(II) phthalocyanine-cobalt(II) tetra(5-phenoxy-10,15,20-triphenylporphyrin), (CoPc-(CoTPP)₄) pentamer as a novel redox mediator for amperometric enzyme electrode sensitive to glucose is described. A glassy carbon electrode (GCE) was first modified with the pentamer, then followed by the immobilization onto the GCE-CoPc-(CoTPP)₄ with glucose oxidase (GOx) through cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) and Nafion^® cation-exchange polymer. The proposed biosensor displayed good amperometric respose charateristics to glucose in pH 7.0 PBS solution; such as low overpotentials (+400 mV versus Ag|AgCl), very fast amperometric response time (∼5 s), linear concentration range extended up to 11 mM, with 10 μM detection limit. The biosensor exhibited electrochemical Michaelis-Menten kinetics and showed an average apparent Michaelis-Menten constant (K^′M) of 14.91 ± 0.46 mM over a storage period of 2 weeks.     

Glucose biosensor based on multiwalled carbon nanotubes grown directly on Si

An amperometric biosensor based on covalent immobilization of glucose oxidase (GOx) on multiwalled carbon nanotubes (MWCNTs) with potassium ferricyanide as the redox mediator was developed. The MWCNTs were grown directly on a layered structure of Co/Ti/Cr on a SiO₂/Si substrate by microwave-heated chemical vapor deposition. The mediator helps to shuttle the electrons between the immobilized GOx and the MWCNT electrode, therefore operating at a potential of 0.25 V vs. the saturated calomel electrode. This potential precludes the interfering compounds from oxidization. The sensitivity of biosensors to glucose was found to depend on the acid pretreatment and GOx reaction times. The steady-state response of the optimized biosensor exhibits a sensitivity of 20.6 μA mM⁻¹ cm⁻², a linear range of up to 8 mM, and a response time of <5 s.     

Dual oxygen and Ir oxide regeneration of glucose oxidase in nanostructured thin film glucose sensors

A nanoparticulate iridium oxide (IrOx) thin film has been developed as a redox-active matrix material for an advanced generation glucose biosensor, in which IrOx serves as the non-physiological mediator, replacing oxygen in the enzymatic re-oxidation of glucose oxidase (GOx). Ethanolic solutions of Nafion and an Ir sol were mixed with an aqueous GOx solution and then deposited on a Au support. The Ir nanoparticles were then oxidized electrochemically to IrOx and the resulting films (IrOx-GOx-Nafion) were tested for their glucose response in both oxygen- and argon-saturated solutions, with the oxygen content in both solutions monitored by a Pt electrode. The sensors that are regenerated largely by O₂ are characterized by a Michaelis-Menten K^′m value of ∼30 mM or more and i_max values of at least 20 μA cm⁻². Under fully deareated conditions, the sensors lose only ∼50% of their response to glucose, clearly indicating that a dual oxygen-regeneration and IrOx mediation mechanism is operative for the biosensor under these conditions. Under optimized conditions, involving a controlled GOx:Ir ratio, only the Ir oxide sites in the film serve to mediate GOx regeneration, giving K^′m (10-15 mM) and i_max values that are independent of the O₂ content of the solution.     

Long-term activity of covalent grafted biocatalysts during intermittent use of a glucose/O2 biofuel cell

The operational stability of enzymes in a concentric glucose/O₂ biofuel cell has been significantly improved with the synthesis of grafted enzyme electrodes compared to entrapped enzyme electrodes. The concentric device combined glucose electro-oxidation by glucose oxidase at the anode and oxygen electro-reduction by bilirubin oxidase at the cathode. The entrapped enzyme electrodes were prepared from physical immobilization of the enzymes by a polypyrrole polymer onto the electrode surface. The grafted enzyme electrodes were synthesized by grafting the enzymes via alkyl spacer arms to a poly(aminopropylpyrrole) film onto the electrode surface. From spectrophotometric and electrochemical analyses, it was demonstrated that the spacer arms increased the operational stability and enzyme mobility that favoured electron transfer from their active sites to the electrode.The maximum power output of the assembled biofuel cell was 20 μW cm⁻², at 0.20 V with 10 mM glucose in phosphate buffer pH 7.4. The grafted enzyme electrodes presented an unprecedented operational stability as the maximum of power density of the BFC remains constant after intermittent use over a 45-day period. This was a remarkable improvement compared to electrodes with entrapped enzymes, which lost 74% of their initial power density after intermittent use over a 17-day period.     

Amperometric bienzyme glucose biosensor based on carbon nanotube modified electrode with electropolymerized poly(toluidine blue O) film

The amperometric bienzyme glucose biosensor utilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) immobilized in poly(toluidine blue O) (PTBO) film was constructed on multi-walled carbon nanotube (MWNT) modified glassy carbon electrode. The HRP layer could be used to analyze hydrogen peroxide with toluidine blue O (TBO) mediators, while the bienzyme system (HRP + GOx) could be utilized for glucose determination. Glucose underwent biocatalytic oxidation by GOx in the presence of oxygen to yield H₂O₂ which was further reduced by HRP at the MWNT-modified electrode with TBO mediators. In the absence of oxygen, glucose oxidation proceeded with electron transfer between GOx and the electrode mediated by TBO moieties without H₂O₂ production. The bienzyme electrode offered high sensitivity for amperometric determination of glucose at low potential, displaying Michaelis-Menten kinetics. The bienzyme glucose biosensor displayed linear response from 0.1 to 1.2 mM with a sensitivity of 113 mA M⁻¹ cm⁻² at an applied potential of −0.10 V in air-saturated electrolytes.     

Bioelectrochemistry and enzymatic activity of glucose oxidase immobilized onto the bamboo-shaped CNx nanotubes

The novel bamboo-shaped CN_x nanotubes, synthesized by nitrogen atoms doping into carbon nanotubes, were used for the immobilization of a relatively large enzyme glucose oxidase (GOx) and its bioelectrochemical studies. The morphologies and adsorptions of GOx immobilization onto CN_x nanotubes were clearly observed by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Electrochemical impedance spectroscopy (EIS) was also used to feature the GOx adsorbed onto the surface of CN_x nanotubes. The immobilized GOx incorporated into CN_x nanotubes films exhibited a well-defined nearly reversible cyclic voltammetric peaks for the electroactive centers of GOx and a fast heterogeneous electron transfer rate with the rate constant (K_s) of 1.96 s⁻¹. The immobilized GOx onto the CN_x nanotubes exhibited its bioelectrocatalytic activity for the oxidation of glucose. The obtained results suggest that with a large amount of defective/active sites on the tube surfaces, a special bamboo structure and a suitable C-N microenvironment introduced by nitrogen doping, CN_x nanotubes could not only facilitate the direct electron transfer between the enzyme and electrode, but also retain the high enzyme loading and the enzymatic bioactivity.     

Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E°′) of −0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k_s) was calculated to be 5.27 s⁻¹. The dependence of E°′ on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.     

Tetracarboxylic acid cobalt phthalocyanine SAM on gold: Potential applications as amperometric sensor for H2O2 and fabrication of glucose biosensor

This report describes the applications of cobalt tetracarboxylic acid phthalocyanine (CoTCAPc) self-assembled monolayer (SAM) immobilized onto a preformed 2-mercaptoethanol (Au-ME) SAM on gold surface (Au-ME-CoTCAPc SAM) as a potential amperometric sensor for the detection of hydrogen peroxide (H₂O₂) at neutral pH conditions. The Au-ME-CoTCAPc SAM sensor showed a very fast amperometric response time of approximately 1 s, good linearity at the studied concentration range of up to 5 μM with a coefficient R² = 0.993 and a detection limit of 0.4 μM oxidatively. Also reductively, the sensor exhibited a very fast amperometric response time (∼1 s), linearity up to 5 μM with a coefficient R² = 0.986 and a detection limit of 0.2 μM. The cobalt tetracarboxylic acid phthalocyanine self-assembled monolayer was then evaluated as a mediator for glucose oxidase (GOx)-based biosensor. The GOx (enzyme) was immobilized covalently onto Au-ME-CoTCAPc SAM using coupling agents: N-ethyl-N(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS), and the results demonstrated a good catalytic behavior. Kinetic parameters associated with the enzymatic and mediator reactions were estimated using electrochemical versions of Lineweaver-Burk and Hanes equation, and the stability of the sensor was tested. The biosensor (Au-ME-CoTCAPc-GOx SAM) electrode showed good sensitivity (7.5 nA/mM) with a good detection limit of 8.4 μM at 3σ, smaller Michaelis-Menten constant (4.8 mM from Hanes plot) and very fast response time of approximately 5 s.     

Development of highly sensitive non-enzymatic sensor for the selective determination of glucose and fabrication of a working model

Copper oxide (CuO)/copper oxalate (CuOx) modified non-enzymatic electrochemical sensor for the detection of glucose in alkaline medium was fabricated by electrochemical anodisation of copper electrodes in potassium oxalate solution. Morphology of the modified copper electrode was studied by Scanning Electron Microscopy (SEM) and its electrochemical behaviour by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). The formation of CuOx on the copper electrode was confirmed by the Infra-red Reflection Absorption Spectrum (IRRAS). The modified electrodes were found to be microporous and rough. Linear Sweep Voltammetry (LSV) and amperometry were adopted to investigate the direct electrocatalytic oxidation of glucose on CuO/CuOx modified electrode in alkaline medium which showed excellent catalytic activity. The best performance of the sensor was obtained at 0.7 V and in 0.1 M sodium hydroxide (NaOH). At this optimum potential, the sensor was highly selective to glucose in the presence of ascorbic acid (AA) and uric acid (UA) which are common interfering species in biological fluids. The sensitivity was found to be very high (1890 μA mM⁻¹ cm⁻²) with excellent linearity (R = 0.9999) up to 15 mM having a low detection limit of 0.05 μM (S/N = 3). The modified electrode was tested for glucose level in blood serum. Based on the optimised conditions, a working model of the sensor was made and successfully tested for glucose.     

Hierarchically assembled ZnO nanosheets microspheres for enhanced glucose sensing performances

Nanostructures with higher surface specific area has great potential applications in sensing devices because higher surface specific area not only improve protein/enzyme immobilization efficiency, but also enhances charge transport and sensing performances. Herein, hierarchically assembled ZnO nanosheets microspheres (HAZNMs) were synthesized by facile one-pot solution process at low-temperature. Results showed that as-synthesized HAZNMs possessing higher specific surface area, significantly increased the enzyme loading efficiency which in turn improved the sensing performances. The as-fabricated biosensors showed a remarkably high sensitivity (210.8 μA/mM cm²) in the wide-linear response range of 0.05–23 mM, favorable stability for long-term storage, excellent anti-interference ability and high reliability for glucose detection in human blood serum samples. The improved sensing performances can be ascribed to the high glucose oxidase (GOx) enzyme immobilization on HAZNMs that provides a favorable microenvironment for the maintenance of GOx enzyme bioactivity.     

A conducting polymer/ferritin anode for biofuel cell applications

An enzyme anode for use in biofuel cells (BFCs) was constructed using an electrically connected bilayer based on a glassy carbon (GC) electrode immobilized with the conducting polymer polypyrrole (Ppy) as electron transfer enhancer, and with horse spleen ferritin protein (Frt) as electron transfer mediator. The surface-coupled redox system of nicotinamide adenine dinucleotide (NADH) catalyzed with diaphorase (Di) was used for the regeneration of NAD⁺ in the inner layer and the NAD⁺-dependent enzyme catalyst glucose dehydrogenase (GDH) in the outer layer. The outer layer of the GC-Ppy-Frt-Di-NADH-GDH electrode effectively catalyzes the oxidation of glucose biofuel continuously; using the NAD⁺ generated at the inner layer of the Di-catalyzed NADH redox system mediated by Frt and Ppy provides electrical communication with enhancement in electron transport. The electrochemical characteristics of the electrodes were investigated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). This anode provides a current density of 1.2 mA cm⁻² in a 45 mM glucose solution and offers a good possibility for application in biofuel cells.     

Alcohol dehydrogenase amperometric biosensor based on a colloidal gold-carbon nanotubes composite electrode

A novel ethanol biosensor based on the bulk incorporation of alcohol dehydrogenase (ADH) into a colloidal gold (Au_coll)-multiwalled carbon nanotubes (MWCNTs) composite electrode using Teflon as binding material is reported. The composite Au_coll-MWCNTs-Teflon electrode exhibited significantly improved electrooxidation of NADH when compared with other carbon composite electrodes, including those based on carbon nanotubes. Amperometric measurements for NADH at +0.3 V showed significant differences in sensitivity between Au_coll-MWCNTs-Teflon and MWCNTs-Teflon composite electrodes. Incorporation of ADH into the bulk electrode material allowed the construction of a mediatorless ethanol biosensor. Both the enzyme loading and the NAD⁺ concentration in solution were optimized. The ADH-Au_coll-MWCNTs-Teflon biosensor allowed a limit of detection for ethanol of 4.7 μmol l⁻¹, which is remarkably better than those reported for other CNTs-based ADH biosensors. The apparent Michaelis-Menten constant was 4.95 mmol l⁻¹, which is much lower than that reported by immobilization of ADH onto a gold electrode. Both repeatability of the ethanol amperometric measurements, reproducibility with different biosensors, lifetime and storage ability can be, in general, advantageously compared with other ADH-CNTs biosensors. The biosensor was applied for the rapid determination of ethanol in commercial and certified beer samples.     

A new amperometric glucose biosensor based on platinum nanoparticles/polymerized ionic liquid-carbon nanotubes nanocomposites

A new amperometric glucose biosensor has been developed based on platinum (Pt) nanoparticles/polymerized ionic liquid-carbon nanotubes (CNTs) nanocomposites (PtNPs/PIL-CNTs). The CNTs was functionalized with polymerized ionic liquid (PIL) through directly polymerization of the ionic liquid, 1-vinyl-3-ethylimidazolium tetrafluoroborate ([VEIM]BF₄), on carbon nanotubes and then used as the support for the highly dispersed Pt nanoparticles. The electrochemical performance of the PtNPs/PIL-CNTs modified glassy carbon (PtNPs/PIL-CNTs/GC) electrode has been investigated by typical electrochemical methods. The PtNPs/PIL-CNTs/GC electrode shows high electrocatalytic activity towards the oxidation of hydrogen peroxide. Taking glucose oxidase (GOD) as the model, the resulting amperometric glucose biosensor shows good analytical characteristics, such as a high sensitivity (28.28 μA mM⁻¹ cm⁻²), wide linear range (up to 12 mM) and low detection limit (10 μM).     

An enzyme-based microfluidic biofuel cell using vitamin K3-mediated glucose oxidation

Viamin K₃-modified poly-l-lysine (PLL-VK₃) was synthesized and used as the electron transfer mediator during catalytic oxidation of NADH by diaphorase (Dp) at the anode of biofuel cell. PLL-VK₃ and Dp were co-immobilized on an electrode and then coated with NAD⁺-dependent glucose dehydrogenase (GDH). The resulting enzymatic bilayer (abbreviated PLL-VK₃/Dp/GDH) catalyzed glucose oxidation. Addition of carbon black (Ketjenblack, KB) into the bilayer enlarged the effective surface area of the electrode and consequentially increased the catalytic activity. An oxidation current of ca. 2 mA cm⁻² was observed when the electrochemical cell contained a stirred 30 mM glucose, 1.0 mM NAD⁺, pH 7.0 phosphate-buffered electrolyte solution. The performance of glucose/O₂ biofuel cells, constructed as fluidic chips with controllable fuel flow and containing a KB/PLL-VK₃/Dp/GDH-coated anode and an Ag/AgCl or a polydimethylsiloxane-coated Pt cathode, were evaluated. The open circuit voltage of the cell with the PDMS-coated Pt cathode was 0.55 V and its maximum power density was 32 μW cm⁻² at 0.29 V when a pH 7.0-buffered fuel containing 5.0 mM glucose and 1.0 mM NAD⁺ was introduced into the cell at a flow rate of 1.0 mL min⁻¹. The cell's output increased as the flow rate increased. During 18 h of continuous operation of the cell with a load of 100 kΩ, the output current density declined by ca. 50%, probably due to swelling of the enzyme bilayer.     

Ni(OH)2 versus Ni/Al layered double hydroxides as matrices to immobilize glucose oxidase

Ni hydroxide and Ni/Al layered double hydroxide (LDH) were electrochemically deposited on Pt electrodes in the presence of glucose oxidase in the electrolytic solution, in order to verify if the performances of the two biosensors were dependent on the Ni content of the inorganic matrix used to entrap the enzyme. The comparative study was conducted by recording the current response due to the oxidation of H₂O₂, produced enzymatically after glucose additions, at +0.45 V versus SCE, pH 7.0 and T = 25 °C.A higher sensitivity and a narrower linearity range were observed for nickel hydroxide based biosensor (up to 5 mM against 15 mM exhibited when LDH was the immobilizing matrix). These results suggested that the amount of the enzyme entrapped on the electrode surface was higher when the matrix was Ni(OH)₂ and they were confirmed by EQCM measurements. On the contrary, the selectivity displayed by the two biosensors in the presence of interfering compounds, such as acetaminophen, citric, uric and ascorbic acids, was almost the same.     

Layer by layer assembly of glucose oxidase and thiourea onto glassy carbon electrode: Fabrication of glucose biosensor

For the first time a novel, simple and facile approach is described to construct highly stable glucose oxidase (GOx) multilayer onto glassy carbon (GC) electrode using thiourea (TU) as a covalent attachment cross-linker. The layer by layer (LBL) attachment process was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and Fourier transform infrared reflection spectroscopy (FT-IR-RS) techniques. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation using ferrocenemethanol as artificial electron transfer mediator and biosensor response was directly correlated to the number of bilayers. The surface coverage of active GOx per bilayer, heterogeneous electron transfer rate constant (k_s) and Michaelis–Menten constant (K_M), of immobilized GOx were 1.50 × 10⁻¹² mol cm⁻², 9.2 ± 0.5 s⁻¹ and 3.42(±0.2) mM, respectively. The biosensor constructed with four-bilayers of TU/GOx showed good stability, high reproducibility, long life-time, fast amperometric response (5 s) with the high sensitivity of 5.73 μA mM⁻¹ cm⁻² and low detection limit of 6 μM at concentration range up to 5.5 mM.     

Aqueous dispersions of reduced graphene oxide and multi wall carbon nanotubes for enhanced glucose oxidase bioelectrode performance

Aqueous dispersions of reduced graphene oxide (rGO) and multi walled carbon nanotubes (MWCNT) were fabricated through a modified chemical reduction method. The significant advantage of the method developed here is the omission of any stabilising compound or organic solvent to obtain stable rGO–MWCNT dispersions. Significantly biological entities, in this case the enzyme glucose oxidase (GOx), can be successfully incorporated into the dispersion. These dispersions were characterised using XPS, SEM, zeta potential and particle size measurements which showed that the dispersion stability is not sacrificed with the addition of GOx, and significantly, the electrical properties of the rGO and MWCNTs are maintained. In this study, rGO acts as an effective dispersing agent for MWCNTs and does not affect the solubility or electroactivity of the GOx. Bioelectrodes fabricated from these rGO–MWCNT–GOx dispersions were characterised electrochemically to test their feasibility in facilitating direct electron transfer (DET) from the redox centre of the enzyme to the electrode. The DET results showed that the specific catalytic current generated at an optimised rGO–MWCNT–GOx electrode was 72 μA/μg GOx, which is 144 times more efficient than other literature values for similar systems. The remarkable specific catalytic current can be attributed to the use of purified enzyme, the efficiency of charge transfer within the rGO–MWCNT composite and the ability of the electrode to facilitate direct electron transfer.