Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC(R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface.     

Automated determination of glucose in soluble coffee using Prussian Blue-glucose oxidase-Nafion((R)) modified electrode

A highly selective, fast and stable biosensor for determination of glucose in soluble coffee has been developed. The biosensor electrode consist of a thin film of ferric hexacyanoferrate (Prussian Blue or PB) electrodeposited on the glassy carbon electrode (GCE) (to provide a catalytic surface for the detection of hydrogen peroxide) glucose oxidase immobilized on top of the electrode and a Nafion^® polymer layer. The stability of the PB film and the biosensor was evaluated by injecting standard-solution (50 μM H₂O₂ and 0.5 mM glucose) during 4 h in a flow-injection system with the electrodes polarized at −50 mV versus Ag/AgCl. The system is able to handle about 60 samples per hour and is very stable and suitable for industrial control. Determination of glucose in the range 2.5 and 15% (w/v) in phosphate buffer with precision (r.s.d. < 1.5%) has been achieved and is in agreement with the conventional procedures. Linear calibration in the range of 0.15 and 2.50 mM with detection limits of ca. 0.03 mM has been obtained. The morphology of the enzyme glucose oxidase on the modified electrode has been analyzed by scanning electron microscopy (SEM) measurements.     

Prussian Blue Modified Amperometric Hg2+ Ion Biosensor Based on Glucose Oxidase Inhibition

In this research a Hg²⁺ ion biosensor was developed by combining Prussian blue (PB) with glucose oxidase (GOx) – an enzyme that can be inhibited by Hg²⁺ ions. An application of PB in the design of Hg²⁺ ion biosensor enabled detecting changes in hydrogen peroxide reduction current at low operational potential of 0.2 vs Ag|AgCl,KCl_sat. The described Hg²⁺ ion biosensor exhibited wide linear range from 27 μM to 247 μM of Hg²⁺ and higher maximal detectable concentration of Hg²⁺ than other GOx inhibition-based biosensors, making it convenient for the analysis of samples with high concentration of Hg²⁺ ions.     

Galactose Oxidase/Prussian Blue Based Biosensors

This paper describes the development of an amperometric biosensor based on galactose oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen‐Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly‐(O‐phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at ?0.2 V vs. Ag‐AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx‐TK biosensor.     

Prussian Blue Nanoparticles Self Assembling on Electrochemically Reduced Graphene Oxide Modified GC Electrode for Sensitive Hydrogen Peroxide Detection

A facile, fast, and convenient route was suggested for the fabrication of Prussian blue nano particles (PBNPs) assembled on reduced graphene oxide (RGO) modified glassy carbon electrode (PBNPs|RGO|GCE). RGO was electrodeposited on the surface of GCE and the prepared RGO|GCE was immersed into a ferric‐hexacyanoferrate(III) solution and PBNPs were assembled on the RGO|GCE for a certain period of time. The PBNPs film thickness can be easily controlled by adjusting the assembling duration. The developed PBNPs|RGO|GCE was successfully used for determining hydrogen peroxide, with a linear response over the concentration range 0.5‐400 μM, a good accuracy and precision, detection limit 0.44 μM, and sensitivity 1168 mA M^?1 cm^?2.     

Prussian blue-glutamate oxidase modified glassy carbon electrode: A sensitive l-glutamate and β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) sensor

A sensitive biosensor has been developed for the neurotoxin β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) contained in the seeds of grass pea (Lathyrus sativus) and for l-glutamate based on glutamate oxidase (GlOx) and a Prussian blue (PB) modified glassy carbon (GC) electrode. The configuration of the system is so as to detect the hydrogen peroxide released during the enzymatic cycle at a low applied potential, −50 mV versus Ag|AgCl, in the flow injection mode. For this purpose GlOx was coupled to PB electrodeposited onto a glassy carbon electrode and stabilised by treatment with tetrabutylammonium toluene-4-sulfonate (TTS) during one of the steps in the electrodeposition. GlOx was cross-linked with glutaraldehyde (GA), bovine serum albumin (BSA) and Tween-20 on the surface of the PB modified GC electrodes. Addition of 0.01% and 0.001% polyethyleneimine (PEI) to the immobilisation mixture resulted in an enhancement of the response signal with about 35% and 62% for glutamate and β-ODAP, respectively, when using 0.01% PEI and with 164% and 200% for glutamate and β-ODAP, respectively, when using 0.001% PEI. The linear response range for β-ODAP was extended from 0.05-0.5 mM to 0.01-1 mM, when 0.001% PEI was used. However, a higher concentration of PEI, 0.1%, caused a decrease in the sensitivity of the biosensor.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Amperometric Glucose Biosensor Based on Glucose Oxidase Encapsulated in Carbon Nanotube–Titania–Nafion Composite Film on Platinized Glassy Carbon Electrode

A highly sensitive and selective glucose biosensor has been developed based on immobilization of glucose oxidase within mesoporous carbon nanotube–titania–Nafion composite film coated on a platinized glassy carbon electrode. Synergistic electrocatalytic activity of carbon nanotubes and electrodeposited platinum nanoparticles on electrode surface resulted in an efficient reduction of hydrogen peroxide, allowing the sensitive and selective quantitation of glucose by the direct reduction of enzymatically‐liberated hydrogen peroxide at ?0.1 V versus Ag/AgCl (3 M NaCl) without a mediator. The present biosensor responded linearly to glucose in the wide concentration range from 5.0×10^?5 to 5.0×10^?3 M with a good sensitivity of 154 mA M^?1cm^?2. Due to the mesoporous nature of CNT–titania–Nafion composite film, the present biosensor exhibited very fast response time within 2 s. In addition, the present biosensor did not show any interference from large excess of ascorbic acid and uric acid.     

Development of biosensors based on hexacyanoferrates

Ferric and copper hexacyanoferrates (PB and CuHCF, respectively) were electrodeposited on glassy carbon electrodes providing a suitable catalytic surface for the amperometric detection of hydrogen peroxide. Additionally glucose oxidase was immobilized on top of these electrodes to form glucose biosensors. The biosensors were made by casting glucose oxidase-Nafion layers onto the surface of the modified electrodes. The operational stability of the films and the biosensors were evaluated by injecting a standard solution (5 muM H(2)O(2) for PB, 5 mM H(2)O(2) for CuHCF and 2.5 mM glucose for both) over 5-10 h in a flow-injection system with the electrodes polarized at -50 (PB) and -200 mV (CuHCF) versus Ag/AgCl, respectively. The glucose biosensors demonstrated suitability for glucose determination: 0.0-2.5 mM (R(2)=0.9977) for PB and 0.0-10 mM (R(2)=0.9927) for CuHCF, respectively. The visualization of the redox catalyst modifiers (PB and CuHCF films) was presented by scanning electron micrographs.     

Prussian Blue Modified Carbon Ionic Liquid Electrode: Electrochemical Characterization and Its Application for Hydrogen Peroxide and Glucose Measurements

Prussian blue modified carbon ionic liquid electrodes (PB‐CILEs) were fabricated using chemical and electrochemical procedures. Chemically fabricated PB‐CILE exhibited an excellent sensitivity (0.0866 μA μM^?1), low detection limit (0.01 μM) and two linear ranges (0.01–1 and 1–600 μM) toward hydrogen peroxide. Then, glucose oxidase (GOx) was immobilized on the surface of PB‐CILE to fabricate glucose biosensor using three different procedures involving cross linking with glutaraldehyde (GLU) and bovine serum albumin (BSA), entrapment into the Nafion matrix and covering with a sol‐gel layer. Glucose biosensor fabricated using cross linking procedure showed the best sensitivity (0.0019 μA μM^?1) and operational stability for glucose.     

Disposable amperometric biosensors based on xanthine oxidase immobilized in the Prussian blue modified screen-printed three-electrode system

The screen-printed three-electrode system was applied to fabricate a new type of disposable amperometric xanthine oxidase biosensor. Carbon-working, carbon-counter and Ag/AgCl reference electrodes were all manually printed on the polyethylene terephthalate substrate forming the screen-printed three-electrode system by the conventional screen-printing process. As a mediator, Prussian blue could not only catalyze the electrochemical reduction of hydrogen peroxide produced from the enzyme reaction, but also keep the favorable potential around 0 V. The optimum operational conditions, including pH, potential and temperature, were investigated. The sensitivities of xanthine and hypoxanthine detections were 13.83 mA/M and 25.56 mA/M, respectively. A linear relationship was obtained in the concentration range between 0.10 μM and 4.98 μM for xanthine and between 0.50 μM and 3.98 μM for hypoxanthine. The small Michaelis-menten constant value of the xanthine oxidase biosensor was calculated to be 3.90 μM. The results indicate that the fabricated xanthine oxidase biosensor is effective and sensitive for the detection of xanthine and hypoxanthine.     

Effect of Various Deposition Techniques,Electrode Materials and Posttreatment with Tetrabutylammonium and Tetrabutylphosphonium Salts on the Electrochemical Behavior and Stability of Various Prussian Blue Modified Electrodes

The effect of various deposition techniques, electrode materials and posttreatment with tetrabutylammonium and tetrabutylphosphonium salts on the electrochemical behavior and stability of various Prussian blue (PB) modified electrodes, namely PB modified glassy carbon electrodes, silicate‐film supported PB modified glassy carbon electrodes, PB‐doped silicate glassy carbon electrodes, PB modified carbon ceramic electrodes using electrochemical deposition and PB modified carbon ceramic electrodes using chemical deposition is reported. Cyclic voltammetry and amperometric measurements of hydrogen peroxide were performed in a flow injection system while the carrier phosphate buffer (pH 7.0) with a flow rate of 0.8 mL min^?1 was propelled into the electrochemical flow through cell housing the PB modified working electrode as well as an Ag|AgCl|0.1 M KCl reference and a Pt auxiliary electrode. The results showed that the deposition procedure, electrode material and posttreatment with additional chemicals can significantly alter the stability and electrochemical behavior of the PB film. Among the studied PB modified electrodes, those based on carbon ceramic electrodes modified with a film of electropolymerized PB showed the best electrochemical stability.     

Highly Sensitive Amperometric Glucose Biosensor Based on Glassy Carbon Electrode with Copper/Palladium Coating

A highly sensitive amperometric glucose biosensor based on immobilizing glucose oxidase in electropolymerized poly(o‐phenylenediamine) film on glassy carbon electrode coated sequentially with copper and palladium layers has been developed. The steady‐state amperometric response to glucose was determined by means of the oxidation of hydrogen peroxide generated by the enzymatic reaction at a potential of either +0.70 or +0.40 V (vs. Ag|AgCl reference). The deposited copper/palladium layer showed great enhancement in the performance of the enzyme electrode, possibly due to its better electrocatalytic activity for hydrogen peroxide oxidation and large surface area. Effects of the relative loading of palladium, enzyme and polymer on the electrode performance were examined in detail. Sensitivity and detection limit for glucose determinations at +0.70 V were about 7.3 μA/mM and 0.1 μM, respectively. A wide linear range up to 6.0 mM glucose could be achieved. Electrode performance was superior to similar works reported in the literature. The response time was less than 2 s and its lifetime was longer than three months. The permeable polyphenylenediamine film also offered good anti‐interference ability to ascorbic acid, uric acid and acetaminophen, especially when a detection potential of +0.40 V was employed.     

普鲁士蓝和苝四甲酸二酐衍生物膜修饰的过氧化氢生物传感器

以玻碳电极(GCE)为基底,采用恒电位法沉积一层普鲁士蓝(PB),然后将苝四甲酸二酐衍生物(PTC-NH2)自组装到其表面,形成既带氨基功能团,又可有效防止PB渗漏的导电膜.通过静电吸附和共价键合作用固定纳米金和辣根过氧化物酶(HRP)的复合物,从而制得性能优良的过氧化氢(H2O2)生物传感器.采用循环伏安法(CV)和计时电流法,考察了传感器的电化学性能.实验表明,本传感器具有灵敏度高、线性范围宽、检出限低、稳定性好、抗干扰能力强等特点.其线性范围为2.0×10-6～1.4×10-3mol/L;检出限为8.3×10-7mol/L(S/N=3).     

Direct Electrochemistry of Glucose Oxidase at Carbon Nanotube‐gold Colloid Modified Electrode with Poly(diallyldimethylammonium chloride) Coating

Glucose oxidase showed direct electrochemical transfer at glassy carbon electrodes immobilized with carbon nanotube‐gold colloid (CNT‐Au) composites with poly(diallydimethylammonium chloride) (PDDA) coatings. The modified electrode (GC/CNT/Au/PDDA‐GOD) was employed for the amperometric determination of glucose. Under optimal conditions, the biosensor displayed linear response to glucose from 0.5 to 5 mM with a sensitivity of 2.50 mA M^?1 at an applied potential of ?0.3 V (vs. Ag|AgCl reference).     

Novel Enzymatic Rhodium Modified Poly(styrene‐g‐oleic amide) Film Electrode for Hydrogen Peroxide Detection

Newly synthesized poly(styrene‐g‐oleic amide) was coated onto a rhodium nanoparticle modified glassy carbon (GC) surface for the fabrication of horseradish peroxidase based biosensor used for hydrogen peroxide detection. The rhodium modifed electrode presented ten times higher signal than unmodified electrode even at low elecrtroactive enzyme quantity by enhancing the electron transfer rate at the applied potential of −0.65 V. The biosensor designed by under the optimized rhodium electrodeposition time exhibited a fast response less than 5 s, an excellent operational stability with a relative standard deviation of 0.6 % (n=6), an accuracy of 96 % and a large linear range between 50 μM and 120 mM for hydrogen peroxide. Detection limit and the sensitivity parameters were calculated to be 44 μM and 57 μA mM⁻¹ cm⁻², respectively by preserving its entire initial response up to the 15 days, while only 20 % of its initial response was lost at the end of one month.     

Osmium Redox Hydrogel Mediated Biosensor for Measurement of Low Concentration Glucose Extracted by Reverse Iontophoresis

An osmium redox hydrogel mediated biosensor for continuous monitoring of glucose extracted from subcutaneous solution by reverse iontophoresis has been developed. For the measurement of low concentration glucose, osmium‐poly(vinylpyridine) wiring horseradish peroxidase was introduced to modify the smooth Au electrodes, and the developed glucose biosensor exhibited a high sensitivity of 11.45 nA μM^?1 cm^?2 and a low detection limit of 2 μM, as well as a high operational stability of more than 97% of its initial activity over a test period of 13.5 h in stirred glucose solution at low applied potential (?0.1 V vs. Ag|AgCl), efficiently inhibiting the electroactive interferences. Permeability of the hydrogels was studied and a diffusion coefficient of 2.4×10^?5 cm²/s for H₂O₂ was obtained. In addition, the effects, such as temperature and the variation happening on Ag|AgCl counter electrode, on determination of glucose were also considered. The proof‐of‐feasibility of the biosensors for the monitoring of the glucose extracted from the subcutaneous solution was tested in vitro, and the responses of the sensors were analyzed. A linear response to current produced by extracted glucose in the concentration range of subcutaneous glucose from 1.0 to 12 mM was obtained with a correlation coefficient up to 0.989. These results testify the feasibility of the developed sensors for measuring the low concentration glucose and have significance for the development of noninvasive glucose monitoring system for the control of diabetes.     

Direct Electrochemistry of Hemoglobin Immobilized on Colloidal Gold‐Hydroxyapatite Nanocomposite for Electrocatalytic Detection of Hydrogen Peroxide

A novel nanocomposite of colloidal gold (GNPs) and hydroxyapatite nanotubes (Hap) was prepared for immobilization of a redox protein, hemoglobin (Hb), on glassy carbon electrode. The immobilized Hb showed fast direct electron transfer and excellent electrocatalytic behavior toward reduction of hydrogen peroxide. A synergic effect between GNPs and Hap for accelerating the surface electron transfer of Hb was observed, which led to a pair of redox peaks with a formal potential of (?340±2) mV at pH 7.0, and a new biosensor for hydrogen peroxide with a linear range from 0.5 to 25 μM and a limit of detection of 0.2 μM at 3σ. Owing to the good biocompatibility of the nanocomposite, the biosensor exhibited good stability and acceptable reproducibility. The as‐prepared nanocomposite film provided a good matrix for protein immobilization and biosensor preparation.     

Anodic Stripping Voltammetric Detection of Arsenic(III) at Gold Nanoparticle‐Modified Glassy Carbon Electrodes Prepared by Electrodeposition in the Presence of Various Additives

The simple, fast and highly sensitive anodic stripping voltammetric detection of As(III) at a gold (Au) nanoparticle‐modified glassy carbon (GC) (nano‐Au/GC) electrode in HCl solution was extensively studied. The Au nanoparticles were electrodeposited onto GC electrode using chronocoulometric technique via a potential step from 1.1 to 0 V vs. Ag|AgCl|NaCl (sat.) in 0.5 M H₂SO₄ containing Na[AuCl₄] in the presence of KI, KBr, Na₂S and cysteine additives. Surfaces of the resulting nano‐Au/GC electrodes were characterized with cyclic voltammetry. The performances of the nano‐Au/GC electrodes, which were prepared using different concentrations of Na[AuCl₄] (0.05–0.5 mM) and KI additive (0.01–1.0 mM) at various deposition times (10–30 s), for the voltammetric detection of As(III) were examined. After the optimization, a high sensitivity of 0.32 mA cm^?2 μM^?1 and detection limit of 0.024 μM (1.8 ppb) were obtained using linear sweep voltammetry.     

Amperometric Glucose Biosensors Based on Integration of Glucose Oxidase onto Prussian Blue/Carbon Nanotubes Nanocomposite Electrodes

Nanosized Prussian blue (PB) particles were synthesized with a chemical reduction method and then the PB nanoparticles were assembled on the surface of multiwall carbon nanotubes modified glassy carbon electrode (PB/MWNTs/GCE). The results showed that the PB/MWNTs nanocomposite exhibits a remarkably improved catalytic activity towards the reduction of hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the PB/MWNTs platform by an electrochemically polymerized o‐phenylenediamine (OPD) film to construct an amperometric glucose biosensor. The biosensor exhibited a wide linear response up to 8 mM with a low detection limit of 12.7 μM (S/N=3). The Michaelis–Menten constant K_m and the maximum current i_max of the biosensor were 18.0 mM and 4.68 μA, respectively. The selectivity and stability of the biosensor were also investigated.