Electrochemical non-enzymatic glucose sensors

The electrochemical determination of glucose concentration without using enzyme is one of the dreams that many researchers have been trying to make come true. As new materials have been reported and more knowledge on detailed mechanism of glucose oxidation has been unveiled, the non-enzymatic glucose sensor keeps coming closer to practical applications. Recent reports strongly imply that this progress will be accelerated in ‘nanoera’. This article reviews the history of unraveling the mechanism of direct electrochemical oxidation of glucose and making attempts to develop successful electrochemical glucose sensors. The electrochemical oxidation of glucose molecules involves complex processes of adsorption, electron transfer, and subsequent chemical rearrangement, which are combined with the surface reactions on the metal surfaces. The information about the direct oxidation of glucose on solid-state surfaces as well as new electrode materials will lead us to possible breakthroughs in designing the enzymeless glucose sensing devices that realize innovative and powerful detection. An example of those is to introduce nanoporous platinum as an electrode, on which glucose is oxidized electrochemically with remarkable sensitivity and selectivity. Better model of such glucose sensors is sought by summarizing and revisiting the previous reports on the electrochemistry of glucose itself and new electrode materials.     

An extremely sensitive monoboronic acid based fluorescent sensor for glucose

An extremely sensitive monoboronic acid based fluorescent sensor for glucose was developed. This was carried out by assembling a fluorescent monoboronic acid, 3-aminophenylboronic acid (PBA) indirectly onto gold surface via its electrostatic interaction with cysteine (Cys) that was directly assembled on the gold surface. The formation of self-assembled bilayers (SAB) was confirmed and primarily characterized by cyclic voltammetry and X-ray photoelectron spectra (XPS). The SAB containing PBA was found fluorescent and its fluorescence showed an extremely high sensitivity to the presence of glucose and other monosaccharides such as galactose and fructose with quenching constants at 10⁸ M⁻¹ order of magnitude compared to those at 10² M⁻¹ in bulk solutions. The quenching constants were found to vary in the order of that is different from that in bulk solution which shows the highest binding affinity toward d-fructose and very low sensitivity toward glucose. The reported monoboronic acid based SAB fluorescent sensor showed the highest sensitivity towards glucose with the capacity of detecting saccharides of concentration down to nanomolar level. It was also demonstrated that the fluorescence from PBA/Cys/Au can be easily recovered after each measurement event and therefore also represents a new reusable method for immobilizing reagent in fabricating chemosensors.     

仿生制备有机-无机复合微囊固定化葡萄糖氧化酶

将层层自组装技术与仿生矿化技术相结合,由聚苯乙烯磺酸钠、聚二甲基二烯丙基氯化铵和二氧化硅成功制备(聚苯乙烯磺酸钠-聚二甲基二烯丙基氯化铵)₂-二氧化硅复合微囊.采用扫描电子显微镜、红外光谱和热重对微囊的形貌和化学结构进行了表征.以该复合微囊作为理想载体固定化葡萄糖氧化酶.结果表明,固定于复合微囊中的葡萄糖氧化酶的热稳定性、pH稳定性、操作稳定性得到了提高;在最适条件下,复合微囊固定化葡萄糖氧化酶的酶活回收率为72.85%,米氏常数是游离葡萄糖氧化酶的2.21倍.复合微囊在化学/生物催化、药物/基因传递系统和生物传感器应用方面具有一定的潜能.     

An electrochemiluminescent sensor for glucose employing a modified carbon nanotube paste electrode

A carbon nanotube paste (CNTP) electrode and a carbon nanotube paste/glucose oxidase (CNTP/GOx) electrode were prepared, and the electrochemiluminescent (ECL) behavior of luminol in the presence of glucose was investigated in detail at each of these electrodes. Compared to the classical carbon paste (CP) electrode, the CNTP electrode incorporating glucose oxidase greatly enhanced the response of the ECL sensor to glucose due to the electrocatalytic activity of the carbon nanotubes, the specificity of the enzymatic reaction, and the sensitivity of the luminol ECL reaction. Under optimal conditions, the electrode was found to respond linearly to glucose in the concentration range 1.0x10(-6) approximately 2.0x10(-3) mol/L, and the detection limit (defined as the concentration that can be detected at a signal-to-noise ratio of 3) was found to be a glucose concentration of 5.0x10(-7) mol/L. The method used to prepare the CNTP/GOx electrode was very convenient, and the electrode surface could be renewed in the case of fouling by simply polishing or cutting it to expose a new and fully active surface. The relative standard deviations (RSD) were found to be 6.8% and 8.9% for the CNTP electrode and the CNTP/GOx electrode (n=6). The electrode retained 95% of its initial response after two weeks.     

Direct electrochemistry of glucose oxidase based on its direct immobilization on carbon ionic liquid electrode and glucose sensing

Direct electrochemistry of glucose oxidase (GOx) has been achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1-butyl pyridinium hexafluophosphate ([BuPy][PF₆]) as binder for the first time. A pair of reversible peaks is exhibited on GOx/CILE by cyclic voltammetry. The peak-to-peak potential separation (ΔE_P) of immobilized GOx is 0.056 V in 0.067 M phosphate buffer solution (pH 6.98) with scan rate of 0.1 V/s. The average surface coverage and the apparent Michaelis–Menten constant are 6.69 × 10⁻¹¹ mol·cm⁻² and 2.47 μM. GOx/CILE shows excellent electrocatalytic activity towards glucose determination in the range of 0.1–800 μM with detection limit of 0.03 μM (S/N = 3). The biosensor has been successfully applied to the determination of glucose in human plasma with the average recoveries between 95.0% and 102.5% for three times determination. The direct electrochemistry of GOx on CILE is achieved without the help of any supporting film or any electron mediator. GOx/CILE is inexpensive, stable, repeatable and easy to be fabricated.     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Electrospun palladium (IV)-doped copper oxide composite nanofibers for non-enzymatic glucose sensors

Pd (IV)-doped CuO oxide composite nanofibers (PCNFs) have been successfully fabricated via electrospinning and then employed to construct an amperometric non-enzymatic glucose sensor. The PCNFs based glucose sensors display distinctly enhanced electrocatalytic activity towards the oxidation of glucose, showing significantly lower overvoltage (0.32 V) and ultrafast (1 s) and ultrasensitive current (1061.4 μA mM⁻¹ cm⁻²) response with a lower detection limit of 1.9 × 10⁻⁸ M (S/N = 3). Additionally, excellent selectivity, reproducibility and stability have also been obtained. These results indicate that PCNFs are promising candidates for amperometric non-enzymatic glucose detection.     

Biocatalytic anode for glucose oxidation utilizing carbon nanotubes for direct electron transfer with glucose oxidase

Covalently linked layers of glucose oxidase, single-wall carbon nanotubes and poly-l-lysine on pyrolytic graphite resulted in a stable biofuel cell anode featuring direct electron transfer from the enzyme. Catalytic response observed upon addition of glucose was due to electrochemical oxidation of FADH₂ under aerobic conditions. The electrode potential depended on glucose concentration. This system has essential attributes of an anode in a mediator-free biocatalytic fuel cell.     

Pd-based nanoporous metals for enzyme-free electrochemical glucose sensors

Nanoporous metals （NPMs） show potential applications as enzyme-free glucose sensors. There are few reports on nanoporous Pd in this area even though their cost is much lower than other NPMs. In this work, we report the formation of Pd-based NPM with improved catalytic activity towards the oxidation of glucose. By dealloying metallic glasses, Pd-based NPMs with hi-continuous networks were obtained. All the Pd-based NPMs show high electrochemical catalytic activity towards glucose oxidation. In this study, NPM with an open, three-dimensional, ligament-channel nanoporous structure resulted by dealloying metallic Pd3oCu4oNiloP2o, producing a pore size of 11 nm and a ligament size of 7 nm as the best configuration towards the direct oxidation reaction of glucose.     

Direct electrochemistry of glucose oxidase entrapped in nano gold particles-ionic liquid-N,N-dimethylformamide composite film on glassy carbon electrode and glucose sensing

The direct electrochemistry of glucose oxidase (GOD) entrapped in nano gold particles (NAs)-N,N-dimethylformamide (DMF)-1-butyl-3-methylimidazolium hexafluophosphate (BMIMPF₆) composite film on a glassy carbon electrode (NAs-DMF-GOD (BMIMPF₆)/GC) has been investigated for first time. The immobilized GOD exhibits a pair of well-defined reversible peaks in 0.050 M pH 5 phosphate solutions (PS), resulting from the redox of flavin adenine dinucleotide (FAD) in GOD. The peak currents are three times as large as those of GOD-NAs-DMF film coated GC electrode (i.e. NAs-DMF-GOD (water)/GC). In addition, the NAs-DMF-GOD (BMIMPF₆) composite material has higher thermal stability than NAs-DMF-GOD (water). Results show that ionic liquid BMIMPF₆, DMF and NAs are requisite for GOD to exhibit a pair of stable and reversible peaks. Without any of them, the peaks of GOD become small and unstable. Upon the addition of glucose, the peak currents of GOD decrease and a new cathodic peak occurs at −0.8 V (versus SCE), which corresponds to the reduction of hydrogen peroxide (H₂O₂) generated by the catalytic oxidation of glucose. The peak current of the new cathodic peak and the glucose concentration show a linear relationship in the ranges of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁶ M and 2.0 × 10⁻⁶ to 2.0 × 10⁻⁵ M. The kinetic parameter I_max of H₂O₂ is estimated to be 1.19 × 10⁻⁶ A and the apparent K_m (Michaelis-Menten constant) for the enzymatic reaction is 3.49 μM. This method has been successfully applied to the determination of glucose in human plasma and beer samples, and the average recoveries are 97.2% and 99%, respectively.     

Oxidation of glucose to gluconic acid by glucose oxidase in a membrane bioreactor

Glucose oxidase (GO) (EC 1.1.3.4) was used as catalyst for oxidizing glucose into gluconic acid utilizing a 10-mL Bioengineering Enzyme Membrane Reactor® or a 400-mL Millipore Stirred Ultrafiltration Cell (MSUC) coupled with a Millipore UF membrane (cutoff of 100 kDa) and operated for 12 h under an agitation of 100 rpm, pH 5.5, and 30°C. The effect of feeding rate (0.10, 0.15, or 0.20 min^?1), glucose (2.5 or 5.0 mM), and GO (1.0 or 2.0 mg/mL) concentrations on the catalysis were studied. A yield of about 75% was attained when the MSUC filled with 1.0 mg/mL of GO was fed with 2.5 mM glucose solution at a rate of 0.15 min^?1.     

Investigation of the origin of the glucose response in a glucose oxidase|polyaniline system

The origin of the signal seen in response to glucose in a polyaniline|glucose oxidase system is explored by immittance spectroscopy, by comparing data from an equivalent circuit model and the parameters obtained from a solution of the faradaic branch of the frequency dispersion for a coupled chemical—electrochemical reaction mechanism. It was shown that an RC subcircuit in the equivalent circuit model was sensitive to peroxide concentration, and the interaction of peroxide with polyaniline at potentials where it either oxidised or reduced the polyaniline was discussed. This information was used to compare the data obtained in a bulk and entrapped glucose oxidaselglucose system, and it was seen that the origin of the response could not be fully attributed to peroxide interaction in the latter case. Under anaerobic conditions with entrapped enzyme, it was proposed that a complex between the gluconolactone product of the enzyme reaction and the polymer leads to a more conducting polymer, with inherent charge compensation, and this results in the observed enhanced current signal.     

Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ_{em max} = 650 nm, λ_{ex max} = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O₂ to produce H₂O₂, which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10⁻⁶–140 × 10⁻⁶ M and a detection limit of 0.7 × 10⁻⁶ M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells.     

New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS)

The immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the water-in-oil (W/O) emulsion system were studied. The terminal amine group was used as the important functionality for GOD immobilization on the silica substrate. When only TEOS is used as a silica source, the disordered mesoporous silica microspheres are obtained. As the molar ratio of APTMS to TEOS (R_AT) increases, the surface area and pore volume of the silica particles measured by nitrogen adsorption and desorption method and SEM decrease rapidly. Particularly, the largest change of the surface morphology is observed between R_AT = 0.20 and R_AT = 0.25. The amount and the adsorption time of immobilized enzyme were measured by UV spectroscopy. About 20 wt% of GOD was immobilized into the silica substrates above R_AT = 0.60 and was completely adsorbed into the substrate of R_AT = 0.80 with lapse of 4 h after addition. In the measurement of the thermal stability, GOD dissolved in buffer solution loses nearly all of its activity after 30 min at 65 °C. In contrast, GOD immobilized on the surface-modified silica particles still retains about 90% of its activity after the same treatment. At this temperature, the immobilized glucose oxidase retained half of its initial activity after 4 h. It is shown that the suitable usage of functionalizing agent like APTMS as well as the control of surface morphology is very important on the immobilization of enzyme.     

Predominating stable adsorption and direct electrochemistry of glucose oxidase on carbon nanotubes by oxygen-containing groups

Stable adsorption and direct electrochemistry of glucose oxidase (GOx) occurred on nitric acid (HNO3)-treated multi-walled carbon nanotubes (MWNTs) instead of as-received MWNTs, demonstrating the critical roles of oxygen-containing groups in stableadsorption and direct electrochemistry of GOx on carbon nanotubes (CNTs).     

Fiber-optic glucose biosensor based on glucose oxidase immobilised in a silica gel matrix

A monolithic silica gel matrix with entrapped glucose oxidase was constructed as a bioactive element in an optical biosensor for glucose determination. Physicochemical and biochemical characterizations of the catalytic matrix were performed, and the intrinsic fluorescence of immobilised glucose oxidase (GOD) was investigated in the UV and visible range by performing steady state and time course measurements. In all cases, the silica gel matrix proved to be a suitable support for optical biosensing owing to its superior optical properties (e.g., high transmittance and reliable fluorescence and GOD absorption spectra after immobilisation). From steady state measurements, calibration curves were obtained as a function of glucose concentration. When time course measurements were performed, the silica gel support displayed a larger linear calibration range and higher sensitivity than other immobilisation systems. In addition, a glucose optical biosensor was developed and characterised using as catalytic element GOD immobilised on a gel disk bound to a bundle of optical fibres.     

Catalysis of the electrochemical oxidation of glucose by glucose oxidase and a single electron cosubstrate: kinetics in viscous solutions

Catalysis of the electrochemical oxidation of glucose by glucose oxidase with a single electron mediator (cosubstrate) may be used to transform mixtures of concentrated industrial sugars. How the high viscosity of such media may affect the enzymatic reaction and the transport of the mediator can be mimicked by addition of large concentrations of sucrose to glucose solutions. Cyclic voltammetry then provides a simple means of investigating the effect of an increased viscosity on the kinetics of the enzymatic reaction and the diffusion of the mediator. The diffusion coefficient of the mediator is decreased 10 times by addition of 1.6 M sucrose. At pH 8, in the presence of the same concentration of sucrose, the catalytic activity of the enzyme towards its substrate is only slightly affected. A 35% decrease of the glucose Michaelis constant is observed. The reaction of the reduced enzyme with the cosubstrate is six times slower and the mediator Michaelis constant undergoes a three-fold increase. It follows that glucose oxidase remains an efficient catalyst in such viscous media.     

A novel amperometric biosensor based on NiO hollow nanospheres for biosensing glucose

NiO hollow nanospheres were synthesized by controlled precipitation of metal ions with urea using carbon microspheres as templates, which were for the first time adopted to construct a novel amperometric glucose biosensor. Glucose oxidase was immobilized on the surface of hollow nanospheres through chitosan-assisted cross-linking technique. Due to the high specific active sites and high electrocatalytic activity of NiO hollow nanospheres, the constructed glucose biosensors exhibited a high sensitivity of 3.43 μA/mM. The low detection limit was estimated to be 47 μM (S/N = 3), and the Michaelis-Menten constant was found to be 7.76 mM, indicating the high affinity of enzyme on NiO hollow nanospheres to glucose. These results show that the NiO hollow nanospheres are a promising material to construct enzyme biosensors.     

Integration of a highly ordered gold nanowires array with glucose oxidase for ultra-sensitive glucose detection

A highly sensitive amperometric nanobiosensor has been developed by integration of glucose oxidase (GO_x) with a gold nanowires array (AuNWA) by cross-linking with a mixture of glutaraldehyde (GLA) and bovine serum albumin (BSA). An initial investigation of the morphology of the synthesized AuNWA by field emission scanning electron microscopy (FESEM) and field emission transmission electron microscopy (FETEM) revealed that the nanowires array was highly ordered with rough surface, and the electrochemical features of the AuNWA with/without modification were also investigated. The integrated AuNWA–BSA–GLA–GO_x nanobiosensor with Nafion membrane gave a very high sensitivity of 298.2 μA cm⁻² mM⁻¹ for amperometric detection of glucose, while also achieving a low detection limit of 0.1 μM, and a wide linear range of 5–6000 μM. Furthermore, the nanobiosensor exhibited excellent anti-interference ability towards uric acid (UA) and ascorbic acid (AA) with the aid of Nafion membrane, and the results obtained for the analysis of human blood serum indicated that the device is capable of glucose detection in real samples.     

Electrodeposited glucose oxidase/anionic clay for glucose biosensors design

An amperometric glucose biosensor was developed using an anionic clay matrix (layered double hydroxide (LDH), Ni/Al-NO₃) for the immobilization of glucose oxidase (GOx). The biofilm was prepared by electrodeposition of the clay and GOx and subsequent cross-linking with glutaraldeyde. The Pt surface modified with the Ni/Al-NO₃ shows a much reduced noise, giving rise to a better signal to noise ratio for the currents relative to H₂O₂ oxidation, and a linear range for H₂O₂ determination wider than the one observed for bare Pt electrodes. Under the optimised operative conditions, the performances of the biosensor have been evaluated by measuring the steady-state currents (at +0.45 V versus SCE) to increasing concentrations of glucose in “air saturated” 0.1 M phosphate buffer (pH 7.0). Both batch and flow injection modes were explored. The response to glucose was linear up to 8.0 and 12.0 mM, and the sensitivities were 7.7 ± 0.1 and 19.1 ± 0.2 mA M⁻¹ cm⁻², respectively. The current response of the biosensors does not significantly change for 15 consecutive days in batch and for 10 days in flow, at least, if stored at 4 °C in phosphate buffer, when not in use. The effects of interferants and applicability to fruit juices and soft drinks analysis of the biosensor were also investigated.