Direct electron transfer of glucose oxidase at glassy carbon electrode modified with functionalized carbon nanotubes within a dihexadecylphosphate film

A glassy carbon electrode modified with functionalized multiwalled carbon nanotubes (CNTs) immobilized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) in a dihexadecylphosphate film was prepared and characterized by cyclic voltammetry and scanning electron microscopy. It was used as a support for FAD or glucose oxidase (GOx) immobilization with EDC/NHS crosslinking agents. Cyclic voltammetry of GOx immobilized onto the surface of CNTs showed a pair of well-defined redox peaks, which correspond to the direct electron transfer of GOx, with a formal potential of −0.418 V vs. Ag/AgCl (3 M KCl) in 0.1 M phosphate buffer solution (pH 7.0). An apparent heterogeneous electron transfer rate constant of 1.69 s⁻¹ was obtained. The dependence of half wave potential on pH indicated that the direct electron transfer reaction of GOx involves a two-electron, two-proton transfer. The determination of glucose was carried out by square wave voltammetry and the developed biosensor showed good reproducibility and stability. The proposed method could be easily extended to immobilize and evaluate the direct electron transfer of other redox enzymes or proteins.     

Amperometric glucose biosensor based on integration of glucose oxidase with platinum nanoparticles/ordered mesoporous carbon nanocomposite

This article reports a new amperometric glucose biosensor based on ordered mesoporous carbon (OMC) supported platinum nanoparticles (Pt/OMC) modified electrode. The Pt/OMC nanocomposite modified electrode exhibited excellent electrocatalytic activities towards the reduction and oxidation of H₂O₂ as well. This feature allowed us to use it as bioplatform on which glucose oxidase (GOD) was immobilized by entrapment in electropolymerized pyrrole film for the construction of the glucose biosensor. The biosensor showed good analytical performances in terms of low detection (0.05 mM), high sensitivity (0.38 μA/mM) and wide linear range (0.05-3.70 mM). In addition, the effects of pH value, applied potential, electroactive interference and the stability of the biosensor were discussed. The applicability to blood analysis was also evaluated.     

A novel photometric glucose biosensor based on decolorizing of silver nanoparticles

A novel glucose biosensor based on chromophore (silver nanoparticles) decolorizing for the photometric determination of glucose was developed. Silver nanoparticles are directly synthesized in the sol-gel matrix by a one-step method based on the reduction of the inorganic precursor AgNO₃ and were used for the preparation, characterization and calibration of a highly sensitive and cost-effective localized surface plasmon resonance-based glucose biosensor. In the presence of glucose oxidase (GOx) and due to the enzyme-substrate (glucose) reaction, H₂O₂ was produced and silver nanoparticles in the sol-gel glass have the ability for the decomposition of hydrogen peroxide. Due to the degradation of silver nanoparticles a remarkable change in the localized surface plasmon resonance absorbance strength could be observed which have been monitored as a suitable signal for determination of substrate concentration. Beer's law is obeyed in the range from 50 to 800 mg/L glucose and the limit of detection is 23 mg/L. The proposed optical biosensor has been successfully applied to the determination of glucose in various real samples.     

An amperometric glucose biosensor based on layer-by-layer GOx-SWCNT conjugate/redox polymer multilayer on a screen-printed carbon electrode

An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0 mM, a high sensitivity of 32 μA mM⁻¹ cm⁻², and a response time of less than 5 s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose.     

Gold nanoparticles-coated eggshell membrane with immobilized glucose oxidase for fabrication of glucose biosensor

This work reports the fabrication and application of a glucose biosensor based on the catalytic effect of gold nanoparticles (AuNPs) on enzymatic reaction for blood glucose determination. AuNPs were initially in situ synthesized on the surface of an eggshell membrane (ESM) which was subsequently immobilized with glucose oxidase (GO_x) to produce a GO_x-AuNPs/ESM. The GO_x-AuNPs/ESM was positioned on the surface of an oxygen electrode to form a GO_x-AuNPs/ESM glucose biosensor. The effects of pH, concentration of phosphate buffer solution and amount of GO_x on the response of the GO_x-AuNPs/ESM glucose biosensor were studied in detail. AuNPs on GO_x/ESM can improve the calibration sensitivity (30% higher than GO_x/ESM without AuNPs), stability (87.3% of its initial response to glucose after 10-week storage) and shortens the response time (<30 s) of the glucose biosensor. The linear working range for the GO_x-AuNPs/ESM glucose biosensor is 8.33 μM to 0.966 mM glucose with a detection limit of 3.50 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose in human blood serum samples and the results compared well to a standard spectrophotometric method commonly used in hospitals. Our work demonstrates that the developed GO_x-AuNPs/ESM glucose biosensor has potential in biomedical analysis.     

One-pot electrodeposition of 3-aminopropyltriethoxysilane-chitosan hybrid gel film to immobilize glucose oxidase for biosensing

We report on the electrodeposition of a 3-aminopropyltriethoxysilane-chitosan (APTES-CS) hybrid gel film for in situ immobilization of glucose oxidase (GOx) on an Au or platinized Au (Pt_nano/Au) electrode for biosensing of glucose. Controllable electroreduction of p-benzoquinone is used to lift the electrode-surface pH for the GOx-APTES-CS codeposition, which was monitored by an electrochemical quartz crystal microbalance. The fabrication procedures of the biosensor and the parameters influencing the biosensing performance were optimized. The prepared porous GOx-APTES-CS/Pt_nano/Au and GOx-APTES-CS/Au electrodes can be used to detect the enzymatically generated H₂O₂ at 0.5 and 0.7 V vs SCE, respectively. The enzyme electrodes exhibited linear responses to glucose concentration from 0.2 μM to 8.2 mM (R = 0.998, at Pt_nano/Au substrate) and from 0.2 μM to 5.5 mM (R = 0.998, at Au substrate), with current sensitivities of 69.5 (Pt_nano/Au) and 65 (Au) μA mM⁻¹ cm⁻², respectively, and a detection limit of 0.2 μM (S/N = 3) was achieved for each electrode. The response time was less than 5 (Pt_nano/Au) or 8 (Au) s. It is striking that the enzyme electrodes remained their initial response sensitivity after storage for 5 (Au) and >6 (Pt_nano/Au) months in 0.10 M PBS (pH 7.0) at 4 °C.     

Low temperature co-fired ceramic (LTCC)-based biosensor for continuous glucose monitoring

This article presents design, fabrication and testing of a miniature ceramic-based biosensor which is destined for continuous glucose monitoring. It is fabricated using well known LTCC (low temperature co-fired ceramics) technology. The biosensor consists of a microreaction chamber, three thick-film electrodes and a microdialysis tube. The detection process is based on oxidation of glucose by molecular oxygen in the presence of enzyme - glucose oxidase, GOx. One of the reaction products is a hydrogen peroxide which is detected amperometrically during its oxidation at a working electrode. Computational fluid dynamics (CFD) analysis was used for optimization of the biosensor geometry and considerations of the influence of operating conditions (flow rate) on mass transfer (diffusion) of the glucose within the microreaction chamber. Tests of the developed LTCC-based biosensor indicate its linear response to glucose concentration up to 9 mM with a relatively high sensitivity of about 147 nA/mM. Moreover, the properties of the presented ceramic biosensor are compared with properties of a similar device made in silicon/glass and in Perspex^®.     

A novel bienzyme glucose biosensor based on three-layer Au-Fe3O4@SiO2 magnetic nanocomposite

The magnetic core-shell Au-Fe₃O₄@SiO₂ nanocomposite was prepared by layer-by-layer assembly technique and was used to fabricate a novel bienzyme glucose biosensor. Glucose oxidase (GOD) and horseradish peroxidase (HRP) were simply mixed with Au-Fe₃O₄@SiO₂ nanocomposite and cross-linked on the ITO magnetism-electrode with nafion (Nf) and glutaraldehyde (GA). The modified electrode was designated as Nf-GOD-HRP/Au-Fe₃O₄@SiO₂/ITO. The effects of some experimental variables such as the pH of supporting electrolyte, enzyme loading, the concentration of the mediator methylene blue (MB) and the applied potential were investigated. The electrochemical behavior of the biosensor was studied using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and chronoamperometry. Under the optimized conditions, the biosensor showed a wide dynamic range for the detection of glucose with linear ranges of 0.05-1.0 mM and 1.0-8.0 mM, and the detection limit was estimated as 0.01 mM at a signal-to-noise ratio of 3. The biosensor exhibited a rapid response, good stability and anti-interference ability. Furthermore, the biosensor was successfully applied to detect glucose in human serum samples, showing acceptable accuracy with the clinical method.     

A glucose oxidase immobilization platform for glucose biosensor using ZnO hollow nanospheres

A good route (template-directed synthetic route) for the fabrication of ZnO hollow nanospheres (ZnO-HNSPs) was proposed. ZnO hollow nanosphere is a wonderful platform to immobilize glucose oxidase for glucose biosensor owing to the high specific surface area and high isoelectric point (IEP). Along with nafion and glucose oxidase (GOD), a glucose sensor was designed. Nafion/ZnO-HNSPs/GOD/GCE displays higher catalytic activity toward the glucose oxidation than Nafion/ZnO nano-Flowers/GOD/GCE. Linear response was obtained over a concentration range from 5.0 × 10⁻³ mM to 13.15 mM with a detection limit of 1.0 μM (S/N = 3), and the sensitivity was 65.82 μA/(mM cm²). Satisfyingly, the Nafion/ZnO-HNSPs/GOD/GCE could effectively avoid the interferences from the common interfering species such as uric acid (UA), ascorbic acid (AA), dopamine (DA) and fructose. The Nafion/ZnO-HNSPs/GOD modified electrode allows high sensitivity, excellently selective, stable, and fast amperometric sensing of glucose and thus is promising for the future development of glucose sensors.     

Analysis of volatile alcohols in apple juices by an electrochemical biosensor measuring in the headspace above the liquid

An electrochemical biosensor was optimised for the analysis of volatile alcohols directly from the gas phase without prior absorption or pre-concentration. The sensor is based on the alcohol oxidase (Pichia pastoris) catalyzed conversion of ethanol and the amperometric detection of the generated hydrogen peroxide. Key part of the three-electrode set-up was a gas-diffusion working electrode (potential: +600 mV vs. Ag/AgCl) that consisted of a porous Teflon membrane coated with a thin platinum layer. Headspace samples were analysed for alcohols and used to derive alcohol concentrations in the liquid phase. The biosensor had a sensitivity of 3.43 μA/mM for ethanol, a response time of 69 s, a linear dynamic range of 0.10-30 mM, a theoretical detection limit (3 < S/N) of 9.9 μM, and a stability of 86% during continuous operation (18 h @ 1 mM ethanol). Using one sensor on three consecutive days, the mean coefficient of variation was 1.3% (three measurements each day @ 10 mM ethanol). Alcohol contents of three apple juices determined with the biosensor were in the range 0.30 g/l-0.67 g/l (equivalent to 6.51 mM-14.5 mM). However, ethanol contents determined by high pressure liquid chromatography coupled to refractive index detection (HPLC-RI) and by a commercial enzyme test kit based on alcohol dehydrogenase ranged from 0.12 g/l to 0.38 g/l (equivalent to 2.60 mM-8.25 mM). Both indicate that the biosensor detected alcohols other than ethanol in the apple juices. HPLC-RI coupled to the biosensor in a flow-through configuration demonstrated that the biosensor detected methanol concomitant to ethanol. Thus, the biosensor could perform a qualitative analysis of the total content of volatile alcohols in apple juices by analysing the gas phase above the sample. This offers the additional advantage that possible, non-volatile interfering substances in the liquid sample cannot impair the measurement.     

Performance characteristic studies of glucose biosensors modified by (3-mercaptopropyl)trimethoxysilane sol–gel and non-conducting polyaniline

The materials (3-mercaptopropyl)trimethoxysilane (MPTMS) sol–gel and non-conducting polyaniline (PAn) were prepared for coating on two different amperometric biosensors fabricated by the immobilization of glucose oxidase (GOx) in conducting polyaniline (PA) as permselective membranes. A few performance characteristics of the MPTMS and the PAn-modified glucose sensors are comparable. In detecting 1 mM glucose, the biosensors’ response times were 11 and 12 s, respectively, and results from the repeating measurements (N = 10) showed that R.S.D. = 2.09 and 1.70%, respectively. The detection linearity of the two biosensors was up to 12.5 mM with R = 0.9989 and 0.9969. When they were used to detect 0.5 mM of ascorbic acid (AA), uric acid (UA), and acetaminophen at an anodic potential of 0.4 V, current signals were either insignificant or invisible. The PAn-coated glucose sensor exhibited superior sensitivity at 18.91 μA/(mM cm²), and a maximum current density of 3.70 mA/cm². GOx in the MPTMS-modified biosensor presented a stronger affinity to glucose with K_m = 48.87. More importantly, the stability of this biosensor is superior to that of the PAn-modified biosensor and has lasted for almost 5 months.     

Conducting polymers with benzothiadiazole and benzoselenadiazole units for biosensor applications

Poly(4,7-di(2,3)-dihydrothienol[3,4-b][1,4]dioxin-5-yl-benzo[1,2,5]thiadiazole) (PBDT) and poly(4,7-di(2,3)-dihydrothienol[3,4-b][1,4]dioxin-5-yl-2,1,3-benzoselenadiazole) (PESeE) were electrochemically deposited on graphite electrodes and used as immobilization matrices for biosensing studies. After electrochemical deposition of the polymeric matrices, glucose oxidase (GOx) was immobilized on the modified electrodes as the model enzyme. In the biosensing studies, the decrease in oxygen level as a result of enzymatic reaction was monitored at −0.7 V vs Ag/AgCl (3.0 M KCl) and correlated with substrate concentration. The biosensor was characterized in terms of several parameters such as operational and storage stabilities, kinetic parameters (K_m and I_max) and surface morphologies. The biosensor was tested on real human blood serum samples.     

层层累积技术制备基于纳米碳管的葡萄糖生物传感器

以多壁纳米碳管(MWCNTs)为电子媒介体和酶的吸附载体,利用层层累积的自组装技术固定葡萄糖氧化酶(GOx)的多层(MWCNTs/GOx)n复合薄膜修饰电极,制备了一种新型葡萄糖生物传感器。结果表明:传感器对葡萄糖的响应电流值随着MWCNTs/GOx复合薄膜层数的不同而变化,当MWCNTs/GOx复合薄膜的层数为6时,响应电流值达到最大。(MWCNTs/GOx)6复合薄膜修饰的葡萄糖生物传感器对3×10-2mol/L葡萄糖的响应电流为1.63μA,响应时间仅为6.7 s。该生物传感器检测的线性范围为5×10-4~1.5×10-2mol/L,最低检测浓度可达0.9×10-4mol/L。     

A stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene)-single walled carbon nanotubes/ascorbate oxidase/nafion films for detection of L-ascorbic acid

In this paper, a stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene) (PEDOT)-single walled carbon nanotubes (SWCNT)/ascorbate oxidase (AO)/Nafion films for detection of l-ascorbic acid (AA) was successfully developed. PEDOT-SWCNT nanocomposite and Nafion films were used as inner and outer films, respectively. AO was immobilized between these two films. The PEDOT-SWCNT nanocomposite films were characterized by electrochemical impedance spectroscopy and scanning electron microscopy. The influence of detection potential and temperature on the biosensor performance was examined in detail. Despite the multilayer configuration, the biosensor exhibited a relatively fast response (less than 10 s) and a linear range from 1 μM to 18 mM (a correlation coefficient of 0.9974). The sensitivity of the biosensor was found to be 28.5 mA M⁻¹ cm⁻². Its experimental detection limit was 0.7 μM (S/N = 3) and the apparent Michaelis-Menten constant (K_m) was calculated to be 18.35 mM. Moreover, the biosensor exhibited good anti-interferent ability and excellent long-term stability. All the results showed that such sandwich-type PEDOT-SWCNT/AO/Nafion films could provide a promising platform for the biosensor designs for AA detection.     

Adsorption of glucose oxidase at platinum-multiwalled carbon nanotube-alumina-coated silica nanocomposite for amperometric glucose biosensor

Glucose oxidase (GOx) has been immobilized in platinum-multiwalled carbon nanotube-alumina-coated silica (Pt-MWCNT-ACS) nanocomposite modified glassy carbon electrode by adsorption to provide a novel amperometric glucose biosensor. The morphology, nature, and performance of the resulting GOx-Pt-MWCNT-ACS nanobiocomposite modified glassy carbon electrode were characterized by field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, cyclic voltammetry, and amperometry. The influence of various experimental conditions was examined for the determination of the optimum analytical performance. The optimized glucose biosensor displayed a wide linear range of up to 10.5 mM, a high sensitivity of 113.13 mA M⁻¹ cm⁻², and a response time of less than 5 s. The sensitivity for the determination of glucose at the GOx-Pt-MWCNT-ACS nanobiocomposite modified glassy carbon electrode is better than at common GOx-Pt-CNT nanobiocomposite modified electrodes. The proposed biosensor has good anti-interferent ability and long-term storage stability after coating with Nafion, and it can be used for the determination of glucose in synthetic serum.     

Potentiometric urea biosensor based on urease immobilized by an electrosynthesized poly(o-phenylenediamine) film with buffering capability

A simple and novel potentiometric biosensor for urea detection was prepared by employing an electrosynthesized polymer with buffering capability. It was obtained by deposition of a weighed amount of urease (Ur) at a glassy carbon (GC) electrode followed by immobilization by an electrosynthesized poly-o-phenylenediamine (PPD) film. An unconventional “upside-down” (UD) geometry was employed for the electrochemical cell. The response of GC/Ur/PPD sensor is linear with urea concentration in the range 10 μM to 1 mM (15 mV/mM, R² = 0.9999) due to buffering capability of PPD film, which represents a novel role of electrosynthesized polymers in their application to biosensors. At higher concentrations, the more common Nernstian response (28 mV/decade, R² = 0.9987) is observed. The sensor exhibits a sufficient sensitivity for practical determinations, rapid response and long term stability.     

Hybridization biosensor based on the covalent immobilization of probe DNA on chitosan-mutiwalled carbon nanotubes nanocomposite by using glutaraldehyde as an arm linker

A label-free DNA biosensor for hybridization detection of short DNA species related to the transgenic plants gene fragment of cauliflower mosaic virus (CaMV) 35S promoter was developed in this paper. The nanocomposite containing chitosan (CS) and mutiwalled carbon nanotubes (MWNTs) was first coated on a glassy carbon electrode. Then a highly reactive dialdehyde reagent of glutaraldehyde (GTD) was applied as an arm linker to covalently graft the 5′-amino modified probe DNA to the CS-MWNTs surface via the facile aldehyde-ammonia condensation reaction. The hybridization capacity of the developed biosensor was monitored with electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as an indicating probe, and the experimental results showed that the biosensor had fast hybridization rate and low background interference. A wide dynamic detection range (1.0 × 10⁻¹³-5 × 10⁻¹⁰ M) and a low detection limit (8.5 × 10⁻¹⁴ M) were achieved for the complementary sequence. In addition, the hybridization specificity experiments showed that the sensing system can accurately discriminate complementary sequence from mismatch and noncomplementary sequences.     

Fabrication of a novel electrochemiluminescence glucose biosensor using Au nanoparticles decorated multiwalled carbon nanotubes

Au nanoparticles (nanoAu) with an average diameter of 60 nm were decorated on the surface of multiwalled carbon nanotubes to prepare MWCNTs-nanoAu nano-hybrids. The MWCNTs-nanoAu nano-hybrids were cast on the surface of a glassy carbon electrode and were then further modified with a layer comprising glucose oxidase and chitosan to fabricate a novel electrochemiluminescence (ECL) glucose biosensor. The biosensor showed a remarkably improved electrocatalytic activity towards luminol oxidation and significant improvement in its ECL response. The proposed ECL biosensor exhibited excellent performance for glucose detection with a wide linear range (1-1000 μM), low detection limit (0.5 μM), excellent reproducibility (0.5%) and satisfactory selectivity.     

Development of a dehydrogenase-based glucose anode using a molecular assembly composed of nile blue and functionalized SWCNTs and its applications to a glucose sensor and glucose/O2 biofuel cell

A simple and new way to immobilize glucose dehydrogenase (GDH) enzyme onto nile blue (NB) covalently assembled on the surface of functionalized single-walled carbon nanotubes (f-SWCNTs) modified glassy carbon (GC) electrode (GDH/NB/f-SWCNTs/GC electrode) was described. The GDH/NB/f-SWCNTs/GC electrode possesses promising characteristics as glucose sensor; a wide linear dynamic range of 100-1700 μM, low detection limit of 0.3 μM, fast response time (1-2 s), high sensitivity (14 μA cm⁻² mM⁻¹), anti-interference ability and anti-fouling. Moreover, the performance of the GDH/NB/f-SWCNTs/GC bioanode was successfully tested in a glucose/O₂ biofuel cell. The maximum power density delivered by the assembled glucose/O₂ biofuel cell could reach 32.0 μW cm⁻² at a cell voltage of 0.35 V with 40 mM glucose. The present procedure can be applied for preparing a potential platform to immobilize different enzymes for various bioelectrochemical applications.     

Selective potentiometric determination of uric acid with uricase immobilized on ZnO nanowires

In this study, a potentiometric uric acid biosensor was fabricated by immobilization of uricase onto zinc oxide (ZnO) nanowires. Zinc oxide nanowires with 80-150 nm in diameter and 900 nm to 1.5 μm in lengths were grown on the surface of a gold coated flexible plastic substrate. Uricase was electrostatically immobilized on the surface of well aligned ZnO nanowires resulting in a sensitive, selective, stable and reproducible uric acid biosensor. The potentiometric response of the ZnO sensor vs Ag/AgCl reference electrode was found to be linear over a relatively wide logarithmic concentration range (1-650 μM) suitable for human blood serum. By applying a Nafion^® membrane on the sensor the linear range could be extended to 1-1000 μM at the expense of an increased response time from 6.25 s to less than 9 s. On the other hand the membrane increased the sensor durability considerably. The sensor response was unaffected by normal concentrations of common interferents such as ascorbic acid, glucose, and urea.