Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Sensor and biosensor preparation, optimisation and applications of Prussian Blue modified electrodes

Being one of the most commonly used electrochemical mediators for analytical applications, Prussian Blue has found a wide use in the biosensor field during the last years. Its particular characteristic of catalysing hydrogen peroxide reduction has been applied in the construction of a large number of oxidase enzyme-based biosensors for clinical, environmental and food analysis. By modifying an electrode surface with Prussian Blue, it is in fact possible to easily detect hydrogen peroxide at an applied potential around 0.0 V versus Ag/AgCl, thus making possible coupling with oxidase enzymes while also avoiding or reducing electrochemical interferences. Papers dealing with glucose, lactate, cholesterol and galactose biosensors that are based on the use of Prussian Blue have recently appeared in the most important analytical chemistry journals. Another recent trend is the use of a choline probe based on choline oxidase for pesticide determination to exploit the inhibition of acetylcholinesterase by these compounds. In addition, the use of Prussian Blue in the development of biosensors for food analysis has captured the interest of many research groups and led to improved methods for the detection of glutamate, galactose, alcohol, fructosyl amine, formate, lysine and oxalate. This review will focus on the biosensing aspects of Prussian Blue-based sensors giving a general overview of the advantages provided by such mediator as well as its drawbacks. A comprehensive bibliographic reference list is presented together with the most up to date research findings in this field and possible future applications. The commercial potential of sensors based on this mediator will also be discussed.     

Electrodeposited nonconducting polytyramine for the development of glucose biosensors

Biosensors with the composition of carbon/Prussian blue/(glucose oxidase+glutaraldehyde+polytyramine) were constructed. Before tyramine monomers were electropolymerized, glucose oxidase and tyramine monomers were cross-linked with glutaraldehyde onto the surface of Prussian-blue-modified electrodes. The constructed biosensors produced highly reproducible and stable devices. The biosensors exhibited neglectable decrease in current response after 10 repeated uses or after 1 month of dry storage. The resultant biosensors had a linear range of 0.1-1 mM glucose and a detection limit of 0.05 mM. Since the following electrocatalytic process proceeds at a low electrode potential (ca. -0.3 V vs Ag/AgCl), ascorbate and uric acid do not produce observable interfering signal for the determination of glucose.     

Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring

Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H(2)O(2)-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency, I(max), K(M)(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H(2)O(2). The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.     

Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 μA mmol⁻¹ l cm^-2 and a limit of detection of 0.20 mmol l⁻¹ being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H₂O₂ produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.     

Development of amperometric biosensor for glucose based on a novel attractive enzyme immobilization matrix: calcium carbonate nanoparticles

Calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 microM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mAcm-2M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.     

Toward continuous glucose monitoring with planar modified biosensors and microdialysis. Study of temperature, oxygen dependence and in vivo experiment

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

Biosensor for the monitoring of hydrogen peroxide using poly(vinyl alcohol) membrane system

Summary A biosensor system for continuous on-line monitoring of hydrogen peroxide concentration was developed employing catalase and a poly(vinyl alcohol)/poly(tetra fluoro ethylene) bilayer membrane system, Catalase was entrapped between poly(vinyl alcohol) membrane layer and poly(tetra fluoro ethylene) membrane layer outside of the galvanic type DO probe. Since poly(vinyl alcohol) membrane has non-porous, hydrophilic characteristics, the difference in hydrogen peroxide concentration between inside and outside of the membrane was therefore approximately 100 times. The developed hydrogen peroxide sensor has a wide linear range of hydrogen peroxide sensing more than 140 mM and favourable dynamic response characteristics. The sensor showed also good operational stability, rapid response time, and long life time.     

The carbon paste electrode encrusted with a microreactor as glucose biosensor.

The amperometric biosensors based on carbon paste electrodes (CPEs) encrusted with single microreactor (MR) have been constructed for the determination of glucose. The MRs were prepared from CPC-silica carrier (CPC) and were loaded with glucose oxidase (GO), mediator (M) and acceptor (A). As the mediator cation radical of 5,10-dimethyl-5, 10-dihydrophenazine (DMDHP), N-methylphenazonium methyl sulfate (PMS) and o-benzoquinone (BQ) and as the acceptor Fe[EDTA]- or Fe(CN)6(3-) was used. The biosensors acted at electrode potential 0.15-0.27 V versus Ag-AgCl electrode. The calibration graphs of the biosensors were linear in the range from 1.5 to 50 mM of glucose. The sensitivity of the biosensors did not change at pH 6-8. The dissolved oxygen little (7%) influenced the biosensors response and 1 mM of ascorbic acid produced the response that was of equal value to 0.5 mM of glucose. The biosensors showed high stability; no change of the response of the biosensors prepared by using the novel microreactor was observed at least for 6 months by keeping the loaded CPC at room temperature in silica container. An optimization of the biosensors response against the GO, the mediator and the polymer amount was performed. The digital modeling of the biosensors action is following.     

Characterization of an organic phase peroxide biosensor based on horseradish peroxidase immobilized in Eastman AQ

Due to their frequent occurrence in food, cosmetics and pharmaceutical products, and their poor solubility in water, the detection of peroxides in organic solvents has aroused significant interest. For diagnostics or on-site testing, a fast and specific experimental approach is required. Although aqueous peroxide biosensors are well known, they are usually not suitable for nonaqueous applications due to their instability. Here we describe an organic phase biosensor for hydrogen peroxide based on horseradish peroxidase immobilized in an Eastman AQ 55 polymer matrix. Rotating disc amperometry was used to examine the effect of the solvent properties, the amount and pH of added buffer, the concentration of peroxide and ferrocene dimethanol, and the amount of Eastman AQ 55 and of enzyme on the response of the biosensor to hydrogen peroxide. The response of the biosensor was limited by diffusion. Linear responses (with detection limits to hydrogen peroxide given in parentheses) were obtained in methanol (1.2 microM), ethanol (0.6 microM), 1-propanol (2.8 microM), acetone (1.4 microM), acetonitrile (2.6 microM), and ethylene glycol (13.6 microM). The rate of diffusion of ferrocene dimethanol was more constrained than the rate of diffusion of hydrogen peroxide, resulting in a comparatively narrow linear range. The main advantages of the sensor are its ease of use and a high degree of reproducibility, together with good operational and storage stability.     

Improved operational stability of chloroperoxidase through use of antioxidants

Chloroperoxidase (CPO) from Caldariomyces fumago is a potentially very useful enzyme due to its ability to catalyze a large variety of stereoselective oxidation reactions, but poor operational stability is a main limitation for commercial use. In the present study, the possibility of increasing the operational stability by use of antioxidants was investigated using the oxidation of indole as model reaction. Caffeic acid was the antioxidant showing the strongest positive effects, reaching a total turnover number (TTN) of 135,000 at pH 4 and 4 mM hydrogen peroxide, compared to 28,700 in the absence of antioxidant. Portion-wise addition of hydrogen peroxide in the presence of caffeic acid caused a further increase in TTN to 171,000. An alternative way to reach high TTN was to use tert-butyl hydroperoxide as oxidant instead of hydrogen peroxide: a TTN of 600,000 was achieved although the reaction was quite slow. In this case, antioxidants did not have any positive effect. Possible mechanisms for the observed inactivation of CPO are discussed.     

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.     

Preparation of gold nanoparticles/functionalized multiwalled carbon nanotube nanocomposites and its glucose biosensing application

Gold nanoparticles stabilized by amino-terminated ionic liquid (Au-IL) have been in situ noncovalently deposited on poly(sodium 4-styrene-sulfonate) (PSS)-functionalized multiwalled carbon nanotubes (MWCNTs) to form a MWCNTs/PSS/Au-IL nanocomposite. PSS can interact with MWCNTs through hydrophobic interaction. Amino-terminated ionic liquid was applied to reduce aqueous HAuCl(4), and the resulting gold nanoparticles were attached to the PSS-functionalized MWCNTs simultaneously. Most gold nanoparticles dispersed well on the functionalized MWCNTs. Transmission electron microscopy, Raman and X-ray photoelectron spectroscopy were used to confirm the composition and structure of the nanocomposites. The resulting MWCNTs/PSS/Au-IL composite exhibits good electrocatalysis toward oxygen and hydrogen peroxide reduction. And good biocompatibility with glucose oxidase was also demonstrated due to its good biocatalysis toward glucose substrate, which offered a friendly environment for the immobilization of biomolecules. Such bionanocomposite provides us potential applications in fabrication of biosensors. The resulting biosensor exhibits good response to glucose with a low detection limit 25 microM. It also has excellent reproducibility, satisfied operational stability and good storage stability.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Pt based enzyme electrode probes assembled with Prussian Blue and conducting polymer nanostructures

Conductive polymer nanotubules of 1,2-diaminobenzene (1,2-DAB) were prepared using a porous polycarbonate membrane template, placed on a Pt foil and used to support the polymer, then, the electropolymerisation was performed by chronocoulometry. The obtained conductive polymer nanostructures were then placed on Pt electrode and used to support highly dispersed prussian blue (PB), which acts as the active component for H2O2 detection. The observed good stability of PB as catalyst of H2O2 was related to the presence of organic non-conventional conducting polymers in a composite nanostructured film. These nanostructured polymer/PB composite films were also characterised by scanning electron microscopy (SEM) and Raman spectroscopy. The non-conventional conducting polymer nanotubules/PB modified Pt electrodes were tested by cyclic voltammeter for stability at different pH values, then, by amperometry, for hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and acetylcholine. Glucose oxidase (GOD), lactate oxidase (LOD), L-amino acid oxidase (L-AAOD), alcohol oxidase (AOD), glycerol-3-phosphate oxidase (GPO), lysine oxidase (LyOx), and choline oxidase (ChOx) were immobilised on PB layer supported on 1,2-diaminobenzene (1,2-DAB) nanotubules onto the Pt electrodes. Different strategies for enzyme immobilisation were performed and used. Analytical parameters such as reproducibility, interference rejection, response time, storage and operational stability of the sensors have been studied and optimised. Results provide a guide to design high sensitive, stable and interference-free biosensors. The glucose biosensors assembled with nanostructured poly(1,2-DAB) showed a detection limit of 5 x 10(-5) mol l(-1), a wide linearity range (5 x 10(-5) to 5 x 10(-3) mol l(-1)), a high selectivity, a stability of 3 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical parameters and stability were also studied for L-(+)-lactic acid, L-leucine, ethanol, glycerol-3-phosphate, lysine, and choline biosensors.     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Extended shelf life of enzyme-based biosensors using a novel stabilization system

The storage stability of amperometric enzyme electrodes has been enhanced by a combination of a soluble, positively charged polymer, diethylaminoethyl (DEAE)-dextran, and a sugar alcohol, lactitol. Two different types of alcohol biosensor have been produced using the enzyme alcohol oxidase, isolated from the methylotrophic yeast Hansenula polymorpha. The first employs enzyme entrapment between two membranes with direct hydrogen peroxide amperometry at +0·65 V. The second was based on the mediated, coupled reaction with horseradish peroxidase and N-methyl phenazimiumtetracyanoquinonedimethane (NMP-TCNQ) on a graphite electrode. In both cases, addition of the stabilizers promoted a considerable increase in the storage stability of the enzyme component, as indicated by an increase in the shelf life of desiccated biosensors under conditions of thermal stress at 37°C. In addition, an L-glutamate biosensor constructed from NMP-TCNQ-modified graphite electrodes and L-glutamate oxidase also exhibited an increase in shelf life when stored, desiccated in the presence of stabilizers.     

Toward real-time continuous brain glucose and oxygen monitoring with a smart catheter

Oxygen and glucose biosensors have been designed, fabricated, characterized and optimized for real-time continuous monitoring on a new smart catheter for use in patients with traumatic brain injury (TBI). Oxygen sensors with three-electrode configuration were designed to achieve zero net oxygen consumption. Glucose sensors were based on the use of platinum nanoparticle-enhanced electrodes that were modified with polycation and glucose oxidase immobilized by chitosan matrix. An iridium oxide electrode was developed to work as a biocompatible reference electrode with enhanced durability and stability in the biological solutions. A study of the effect of temperature on oxygen sensor performance, and both temperature and oxygen effects on glucose sensor performance were accomplished to enhance their operative stability and provide useful information for in vivo applications. A new methodology for automatic correction of the temperature and oxygen dependence of biosensor outputs is demonstrated through programmed LabView™ software. In vitro experiments in both physiological and pathophysiological ranges (oxygen: 0–60 mmHg; glucose: 0.1–10 mM; temperature: 25–40 °C) with clinical samples of cerebrospinal fluid obtained from TBI patients have demonstrated stable measurements with enhanced accuracy, indicating the feasibility of the sensors for a real-time continuous in vivo monitoring.