Indirect electrocatalytic determination of choline by monitoring hydrogen peroxide at the choline oxidase-prussian blue modified iron phosphate nanostructures

Choline, as a marker of cholinergic activity in brain tissue, is very important in biological and clinical analysis, especially in the clinical detection of the neurodegenerative disorders disease. This work presents an electrochemical approach for the detection of choline based on prussian blue modified iron phosphate nanostructures (PB-FePO(4)). The obtained nanostructures showed a good catalysis toward the electroreduction of H(2)O(2), and an amperometric choline biosensor was developed by immobilizing choline oxidase on the PB-FePO(4) nanostructures. The biosensor exhibited a rapid response (ca. 2s), low detection limit (0.4±0.05 μM), wide linear range (2 μM to 3.2 mM), high sensitivity (~75.2 μAm M(-1) cm(-2)), as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid and 4-acetamidophenol did not cause obvious interference due to the low detection potential (-0.05 V versus saturated calomel electrode). This nanostructure could be used as a promise platform for the construction of other oxidase-based biosensors.     

A screen-printed microband glucose biosensor system for real-time monitoring of toxicity in cell culture

Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (E(app) of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24 h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10 mM (dynamic range 30 mM). Within the linear region, a correlation coefficient (R(2)) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24 h, an estimated 0.15% (89 nmol) of the starting glucose concentration (24 mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4. Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 10(6) cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1 mM paracetamol, glucose uptake was inhibited by 95% after 6 h in culture; at 500 μM, around 15% inhibition was observed after 16 h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system.     

Layer-by-layer self-assembly of functionalized graphene nanoplates for glucose sensing in vivo integrated with on-line microdialysis system

In this work, a novel amperometric biosensor for hydrogen peroxide was fabricated through the layer-by-layer (LBL) self-assembling of amine-terminated ionic liquid (IL-NH(2)), and sulfonic acid (SO(3)(-)) functionalized graphene by covalent bonding. The modification of the two functionalities introduced positive and negative charge onto the surface of graphene respectively, thus facilitating the formation of a multilayer film denoted with {IL-RGO/S-RGO}(n) through electrostatic interaction and further immobilization of glucose oxidase (GOx). The resulting {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor displayed an excellent response to glucose at a potential of -200 mV. Combined with on-line microdialysis system, the glucose biosensor in the on-line system showed good linear range from 10 μM to 500 μM with the detection limit of 3.33 μM (S/N=3). Consequently, the basal level of glucose in the striatum of anesthetic rats was calculated to be 0.376 ± 0.028 mM (mean ± s.d., n=3). The {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor was further applied for in vivo sensing of the glucose level in the striatum when rats received intraperitoneal (i.p.) injection of 30 μL insulin, which resulted in an obvious decrease in the extracellular concentration of glucose within 30 min. The method was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.     

Glucose biosensor based on nanohybrid material of gold nanoparticles and glucose oxidase on a bioplatform

A simple and relatively cheap glucose biosensor based on a combination of gold nanoparticles (Au NPs) and glucose oxidase (GO(x) ) immobilized on a bioplatform eggshell membrane was established. Scanning electron microscopy showed successful immobilization of Au NPs/GO(x) on the eggshell membrane. The effects of pH, phosphate buffer concentration, and temperature on the glucose biosensor were studied in detail. The biosensor shows a linear response at a glucose concentration range of 5-525 μM. The detection limit of the biosensor is 2.5 μM (S/N = 3). The biosensor exhibits good repeatability with RSD = 3.6% (n = 6), good operational stability with over 300 measurements and long-term storage stability with a shelf life of at least 6 months. The response time is less than 60 s. The glucose level in commercial food samples has been successfully determined. The proposed work shows potential to develop cost-effective biosensors for biotechnological, biomedical and industrial use.     

Amperometric glucose biosensor utilizing FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode

Amperometric glucose biosensors utilizing commercially available FAD-dependent glucose dehydrogenases from two strains of Aspergillus species are described. Enzymes were immobilized on nanocomposite electrode consisting of multi-walled carbon nanotubes by entrapment between chitosan layers. Unlike the common glucose oxidase based biosensor, the presented biosensors appeared to be O(2)-independent. The optimal amount of enzymes, working potential and pH value of working media of the glucose biosensors were determined. The biosensor utilizing enzyme isolated from Aspergillus sp. showed linearity over the range from 50 to 960 μM and from 70 to 620 μM for enzyme from Aspergillus oryzae. The detection limits were 4.45 μM and 4.15 μM, respectively. The time of response was found to be 60 s. The biosensors showed excellent operational stability - no loss of sensitivity after 100 consecutive measurements and after the storage for 4 weeks at 4 °C in phosphate buffer solution. When biosensors were held in a dessicator at room temperature without use, they kept the same response ability at least after 6 months. Finally, the results obtained from measurements of beverages and wine samples were compared with those obtained with the enzymatic-spectrophotometric and standard HPLC methods, respectively. Good correlation between results in case of analysis of real samples and good analytical performance of presented glucose biosensor allows to use presented concept for mass production and commercial use.     

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.     

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 μM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 μM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 μM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 μM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 μM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 μM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate.     

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.     

Electrochemical biosensing utilizing synergic action of carbon nanotubes and platinum nanowires prepared by template synthesis

Platinum nanowires (PtNWs) prepared by electrodeposition method with the help of porous anodic aluminum oxide (AAO) templates have been solubilized in chitosan (CHIT) together with carbon nantubes (CNTs) to form a PtNW-CNT-CHIT organic-inorganic system. The resulting PtNW-CNT-CHIT material brings capabilities for utilizing synergic action of PtNWs and CNTs to facilitate electron-transfer process in electrochemical sensor design. The PtNW-CNT-CHIT film modified electrode offered a significant decrease in the overvoltage for the hydrogen peroxide and showed to be excellent amperometric sensors for hydrogen peroxide at -0.1 V over a wide range of concentrations, and the sensitivity is 260 microAmM-1cm-2. As an application example, by linking glucose oxidase (GOx), an amplified biosensor toward glucose was prepared. The glucose biosensor exhibits a selective determination of glucose at -0.1 V with a linear response range of 5 x 10(-6) to 1.5 x 10(-2)M with a correlation coefficient of 0.997, and response time <10s. The high sensitivity of the glucose biosensor is up to 30 microAmM-1cm-2 and the detection limit was 3 microM. The biosensor displays rapid response and expanded linear response range, and excellent repeatability and stability.     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Self-assembled graphene platelet-glucose oxidase nanostructures for glucose biosensing

Graphene platelet-glucose oxidase (GP-GOD) nanostructures have been prepared through self-assembly of GOD and chitosan (CS) functionalized GPs by electrostatic attraction in aqueous solution. The stable aqueous dispersion of GPs was prepared by chemical reduction of graphene oxide with the use of CS as a reducing and stabilizing agent. UV-vis spectroscopy, X-ray diffraction, transmission electron microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the resulting GPs and GP-GOD nanostructures. Furthermore, a glucose biosensor was constructed by deposition of the resultant GP-GOD on the surface of glassy carbon electrode. It was found that the resulting biosensor exhibits good response to glucose. The linear detection range is estimated to be from 2 to 22 mM (r=0.9987), and the detection limit is estimated to be 20 μM at a signal-to-noise ratio of 3.     

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.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

In situ synthesis of palladium nanoparticle-graphene nanohybrids and their application in nonenzymatic glucose biosensors

A nonenzymatic electrochemical biosensor was developed for the detection of glucose based on an electrode modified with palladium nanoparticles (PdNPs)-functioned graphene (nafion-graphene). The palladium nanoparticle-graphene nanohybrids were synthesized using an in situ reduction process. Nafion-graphene was first assembled onto an electrode to chemically adsorb Pd(2+). And Pd(2+) was subsequently reduced by hydrazine hydrate to form PdNPs in situ. Such a PdNPs-graphene nanohybrids-based electrode shows a very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium. The proposed biosensor can be applied to the quantification of glucose with a wide linear range covering from 10 μM to 5mM (R=0.998) with a low detection limit of 1 μM. The experiment results also showed that the sensor exhibits good reproducibility and long-term stability, as well as high selectivity with no interference from other potential competing species.     

Piezoelectric detection of bilirubin based on bilirubin-imprinted titania film electrode

A novel quartz crystal microbalance (QCM) sensor with a high selectivity and sensitivity has been developed for bilirubin determination, based on the modification of bilirubin-imprinted titania film onto a quartz crystal by molecular imprinting and surface sol-gel techniques. The performance of the developed bilirubin biosensor was evaluated and the results indicated that a sensitive bilirubin biosensor could be fabricated. The obtained bilirubin biosensor presents high-selectivity monitoring of bilirubin, better reproducibility, shorter response time (30 min), wider linear range (0.1-50 μM), and lower detection limit (0.05 μM). The analytical application of the bilirubin biosensor confirms the feasibility of bilirubin determination in serum sample.     

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.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

Nanoflake-like SnS₂ matrix for glucose biosensing based on direct electrochemistry of glucose oxidase

A novel biosensor is developed based on immobilization of proteins on nanoflake-like SnS? modified glass carbon electrode (GCE). With glucose oxidase (GOD) as a model, direct electrochemistry of the GOD/nanoflake-like SnS? is studied. The prepared SnS? has large surface area and can offer favorable microenvironment for facilitating the electron transfer between protein and electrode surface. The properties of GOD/SnS? are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry (CV), respectively. The immobilized enzyme on nanoflake-like SnS? retains its native structure and bioactivity and exhibits a surface-controlled, reversible two-proton and two-electron transfer reaction with the apparent electron transfer rate constant (k(s)) of 3.68 s?1. The proposed biosensor shows fast amperometric response (8s) to glucose with a wide linear range from 2.5 × 10?? M to 1.1 × 10?3 M, a low detection limit of 1.0 × 10?? M at signal-to-noise of 3 and good sensitivity (7.6 ± 0.5 mA M?1 cm?2). The resulting biosensor has acceptable operational stability, good reproducibility and excellent selectivity and can be successfully applied in the reagentless glucose sensing at -0.45 V. It should be worthwhile noting that it opens a new avenue for fabricating excellent electrochemical biosensor.     

A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment

A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrile-co-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (< 30s) with a linear range of 5 x 10(-6) to 3 x 10(-3)M and a sensitivity of 6.82 mAM(-1)cm(-2). The apparent Michaelis-Menten constant (K(M)(app)) was 7.3mM.