Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

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.     

Disposable biosensor based on enzyme immobilized on Au-chitosan-modified indium tin oxide electrode with flow injection amperometric analysis

Indium tin oxide (ITO) electrode is used to fabricate a novel disposable biosensor combined with flow injection analysis for the rapid determination of H2O2. The biosensor is prepared by entrapping horseradish peroxidase (HRP) enzyme in colloidal gold nanoparticle-modified chitosan membrane (Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanning electron microscope, atomic force microscope, and electrochemical methods. Parameters affecting the performance of the biosensor, including concentrations of o-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under the optimal experimental conditions, H2O2 could be determined in the linear calibration range from 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n=8). The amperometric response of the biosensor did not show an obvious decrease after the substrates were injected continuously 34 times into the flow cell. The prepared biosensor not only is economic and disposable, due to the low-cost ITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication in batch.     

Development of a sandwich format, amperometric screen-printed uric acid biosensor for urine analysis

A screen-printed carbon electrode (SPCE) incorporating the electrocatalyst cobalt phthalocyanine (CoPC), fabricated using a water-based ink formulation, has been investigated as the base transducer for a uric acid biosensor. A sandwich biosensor was fabricated by first depositing cellulose acetate (CA) onto this transducer (CoPC-SPCE), followed by uricase (UOX) and finally a polycarbonate (PC) membrane; this device is designated PC-UOX-CA-CoPC-SPCE. This biosensor was used in conjunction with chronoamperometry to optimize the conditions for the analysis of urine: temperature, 35°C; buffer, pH 9.2; ionic strength, 50 mM; uricase, 0.6 U; incubation time, 180 s. The proposed biosensor was applied to urine from a healthy subject. The precision determined on unspiked urine (n=6) was 5.82%. Urine was fortified with 0.225 mM UA, and the resulting precision and recovery were 4.21 and 97.3%, respectively. The linear working range of the biosensor was found to be 0.015 to 0.25 mM (the former represents the detection limit), and the sensitivity was calculated to be 2.10 μA/mM.     

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.     

Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite

A novel amperometric biosensor for hydrogen peroxide (H(2)O(2)) was developed by entrapping horseradish peroxidase (HRP) in a new ormosil composite doped with ferrocene monocarboxylic acid-bovine serum albumin conjugate and multiwall carbon nanotubes (MWNTs). The ormosil was prepared using 3-(aminopropyl)triethoxysilane and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers. The encapsulated conjugate showed excellent electrochemistry and acted as an electron transfer mediator. The presence of MWNTs improved the conductivity of the composite film. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H(2)O(2). The proposed H(2)O(2) biosensor exhibited a linear range of 0.02-4.0 mM with a detection limit of 5.0 microM (S/N = 3) and a K(M)(app) value of 2.0 mM. It could be used for flow injection analysis of hydrogen peroxide with a liner range from 0.02 to 4.5 mM, sensitivity of 0.042 microA/mM and analytical time of 20 s per sample. This biosensor possessed good analytical performance and storage stability.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Na?on film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062-14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM(-1)cm(-2)). The Michaelis-Menten constant (K(m)) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.     

Amperometric phenol biosensor based on laponite clay-chitosan nanocomposite matrix

A novel strategy to fabricate an amperometric biosensor for phenol determination based on chitosan/laponite nanocomposite matrix was described. The composite film was used to immobilize PPO on the surface of a glassy carbon electrode. Chitosan was utilized to improve the analytical performance of the pure clay-modified bioelectrode. The biosensor exhibited a series of properties: good affinity to its substrate (the apparent Michaelis-Menten constant for the sensor was found to be 0.16 mM), high sensitivity (674 mA M(-1)cm(-2) for catechol) and remarkable long-term stability in storage (it retains 88% of the original activity after 60 days). In addition, optimization of the biosensor construction as well as effects of experimental variables such as pH, operating potential and temperature on the amperometric response of the sensor were discussed.     

Extradiol dioxygenase-SiO₂ sol-gel modified electrode for catechol and its derivatives detection

A feasible and sensitive biosensor for catechol and its derivatives using 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)-modified glassy carbon electrode was successfully constructed by polyvinyl alcohol-modified SiO? sol-gel method. The as-prepared biosensor was characterized by electrochemical impedance spectroscopy, and the surface topography of the film was imaged by atomic force microscope. Liquid chromatography-tandem mass spectrometry was applied to reveal the catalytic mechanism. BphC embedded in SiO? gel maintained its bioactivity well and exhibited excellent eletrocatalytical response to both catechol and some of its derivatives (such as 3-methylcatechol and 4-methylcatechol). The biosensor showed a linear amperometric response range between 0.002 mM and 0.8 mM catechol. And the sensitivity was 1.268 mA/(mM cm2) with a detection limit of 0.428 μM for catechol (S/N = 3). Furthermore, the BphC biosensor exhibited perfect selectivity for catechol in the mixtures of catechol and phenol. It was suggested that this flexible protocol would open up a new avenue for designing other ring-cleavage enzyme biosensors, which could be widely used for monitoring various kinds of environmental pollutants.     

Supramolecular architecture based on the self-assembling of multiwall carbon nanotubes dispersed in polyhistidine and glucose oxidase: Characterization and analytical applications for glucose biosensing

We report for the first time the development of a sensitive and selective glucose biosensor based on the self-assembling of multiwall carbon nanotubes (MWCNTs) dispersed in polyhistidine (Polyhis) and glucose oxidase (GOx) on glassy carbon electrodes (GCE). The supramolecular architecture was characterized by SEM, FT-IR and electrochemical techniques. The optimum multistructure was obtained with five (MWCNT-Polyhis/GOx) bilayers and one layer of Nafion as anti-interferent barrier. The sensitivity at 0.700V was (1.94±0.03) mAM(-1) (r=0.9991), with a linear range between 0.25 and 5.00mM, a detection limit of 2.2μM and a quantification limit of 6.7μM with minimum interference from lactose (1.5%), maltose (5.7%), galactose (1.2%), ascorbic acid (1.0%), and uric acid (3.3%). The biocatalytic layer demonstrated to be highly reproducible since the R.S.D. for 10 successive amperometric calibrations using the same surface was 3.6%. The sensitivity of the biosensor after 15 day storage at 4°C remained at 90% of its original value. The combination of the excellent dispersing properties and polycationic nature of polyhistidine, the stability of the MWCNT-Polyhis dispersion, the electrocatalytic properties of MWCNTs, the biocatalytic specificity of GOx, and the permselective properties of Nafion have allowed building up a sensitive, selective, robust, reproducible and stable glucose amperometric biosensor for the quantification of glucose in milk samples.     

Selective determination of dopamine with an amperometric biosensor using electrochemically pretreated and activated carbon/tyrosinase/Nafion®-modified glassy carbon electrode

Dopamine, the most important neurotransmitter in the human brain, controls various functions. Dopamine deficiency causes fatal neurological disorders such as Parkinson’s disease. Even though various types of electrochemical sensors have been studied to measure dopamine levels, they often have poor selectivity for dopamine due to co-existence of interfering substances (e.g. ascorbic acid). Herein, we aimed to develop a highly sensitive dopamine detection method in the co-existence of ascorbic acid, a major interfering substance in real sample by designing an electrochemically pretreated and activated carbon/tyrosinase/Nafion®-modified GCE as an amperometric dopamine biosensor. To maximize the biosensor performance, pH, volume of Nafion®, and scan rate were optimized. This electrochemically pretreated and activated carbon/tyrosinase/ Nafion®-modified GCE could detect as low as 50 μM of dopamine with a wide linear range (50 ~ 1,000 μM) within a few seconds. In addition, it had a sensitivity of 103mAM/cm², which was higher than all previously reported tyrosinasebased dopamine biosensors. In addition, interference effect caused by 4 mM of ascorbic acid was negligible in the co-existence of 1 mM of dopamine. Consequently, this electrochemically pretreated and activated carbon/tyrosinase/ Nafion®-modified GCE might be applicable as amperometric biosensor for selective detection of dopamine in real samples with interfering substances.     

Properties of modified amperometric biosensors based on methanol dehydrogenase and cells Methylobacterium nodulans

The properties of amperometric biosensors based on methanol dehydrogenase (MDH) Methylobacterium nodulans, cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition of hydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135–0.5 and 0.032–1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 μM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.     

Application of hydrophobic palladium nanoparticles for the development of electrochemical glucose biosensor

An amperometric glucose biosensor based on an n-alkylamine-stabilized palladium nanoparticles (PdNPs)-glucose oxidase (GOx) modified glassy carbon (GC) electrode has been successfully fabricated. PdNPs were initially synthesized by a biphase mixture of water and toluene method using n-alkylamines (dodecylamine, C??-NH? and octadecylamine, C??-NH?) as stabilizing ligands. The performance of the PdNPs-GOx/GC biosensor was studied by cyclic voltammetry. The optimum working potential for amperometric measurement of glucose in pH 7.0 phosphate buffer solution is -0.02 V (vs. Ag/AgCl). The analytical performance of the biosensor prepared from C??-PdNPs-GOx is better than that of C??-PdNPs-GOx. The C??-PdNPs-GOx/GC biosensor exhibits a fast response time of ca. 3s, a detection limit of 3.0 μM (S/N=3) and a linear range of 3.0 μM-8.0 mM. The linear dependence of current density with glucose concentration is 70.8 μA cm?2 mM?1. The biosensor shows good stability, repeatability and reproducibility. It has been successfully applied to determine the glucose content in human blood serum samples.     

Construction of an amperometric glucose biosensor based on the immobilization of glucose oxidase onto electrodeposited Pt nanoparticles-chitosan composite film

One-step construction of Pt nanoparticles-chitosan composite film (PtNPs-CS) was firstly proposed as a novel immobilization matrix for the enzymes to fabricate glucose biosensor. This novel interface embedded in situ PtNPs in CS hydrogel was developed by one-step electrochemical deposition in solution containing CS and chloroplatinic acid (H(2)PtCl(6)). Several techniques, including scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry were employed to characterize the assembly process and performance of the biosensor. Under the optimized experimental conditions, the resulting biosensor exhibited excellent linear behavior in the concentration range from 1.2 μM to 4.0 mM for the quantitative analysis of glucose with a limit of detection of 0.4 μM at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 2.4 mM, showing good affinity. The proposed biosensor offered good amperometric responses to glucose due to the nanostructured sensing film provided plenty of active sites for the immobilization of glucose oxidase (GOD).     

Characteristics of third-generation glucose biosensors based on Corynascus thermophilus cellobiose dehydrogenase immobilized on commercially available screen-printed electrodes working under physiological conditions

In this article, we describe a third-generation amperometric glucose biosensor working under physiological conditions. This glucose biosensor consists of a recently discovered cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) immobilized on different commercially available screen-printed electrodes made of carbon (SPCEs), carboxyl-functionalized single-walled carbon nanotubes (SPCE-SWCNTs), or multiwalled carbon nanotubes (SPCE-MWCNTs) by simple physical adsorption or a combination of adsorption followed by cross-linking using poly(ethyleneglycol) (400) diglycidyl ether (PEGDGE) or glutaraldehyde (GA). The CtCDH-based third-generation glucose biosensor has a linear range between 0.025 and 30 mM and a detection limit of 10 μM glucose. Biosensors based on SWCNTs showed a higher sensitivity and catalytic response than the ones functionalized with MWCNTs and the SPCEs. A drastic increase in response was observed for all three electrodes when the adsorbed enzyme was cross-linked with PEGDGE or GA. The operational stability of the biosensor was tested for 7 h by repeated injections of 50 mM glucose, and only a slight decrease in the electrochemical response was found. The selectivity of the CtCDH-based biosensor was tested on other potentially interfering carbohydrates such as mannose, galactose, sucrose, and fucose that might be present in blood. No significant analytical response from any of these compounds was observed.     

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.     

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.