Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

Sensitive amperometric biosensor for the determination of biogenic and synthetic amines using pea seedlings amine oxidase: a novel approach for enzyme immobilisation

We prepared a new inorganic sorbent based on modified triazine (2-[4,6-bis (aminoethylamine)-1,3,5-triazine]-Silasorb; BAT-Silasorb) which binds pea seedlings amine oxidase (PSAO) very tightly without loss of its catalytic activity. This unique feature as well as the wide substrate specificity of PSAO was successfully utilised in the construction of an amperometric biosensor based on a carbon paste electrode for the fast and sensitive detection of various amines at a formal potential 0 mV versus Ag/AgCl reference electrode. The reaction layer of the biosensor is created by the direct immobilisation of PSAO at the electrode surface via affinity carrier BAT-Silasorb. Used arrangement facilitates a simple restoration of the inactive biosensor. An amperometric signal results from horseradish peroxidase catalysed reduction of H2O2, a secondary product of the oxidative deamination of amines, catalysed by PSAO. The sensor was used for the basic characterisation of 55 biogenic and synthetic amines, from numerous mono-, di- and polyamines to various hydroxy-, thio-, benzyl- and aromatic derivatives in order to establish its suitability as a postcolumn detector. Its high sensitivity to putrescine 20.0 +/- 0.64 mA l-1 per mol (636.9 +/- 2.03 mA l-1 per mol per cm2), a limit of detection of 10 nmol l-1 (determined with respect to a signal-to-noise ratio 3:1), a linear range of current response to 0.01-100 mumol l-1 concentration of substrate and good reproducibility all indicate that the sensor could be applied to future industrial and clinical analyses.     

Glucose biosensor based on Au nanoparticles-conductive polyaniline nanocomposite

In this paper, we report a novel glucose biosensor based on composite of Au nanoparticles (NPs)-conductive polyaniline (PANI) nanofibers. Immobilized with glucose oxidase (GOx) and Nafion on the surface of nanocomposite, a sensitive and selective biosensor for glucose was successfully developed by electrochemical oxidation of H2O2. The glucose biosensor shows a linear calibration curve over the range from 1.0x10(-6) to 8.0x10(-4) mol/L, with a slope and detection limit (S/N=3) of 2.3 mA/M and 5.0x10(-7) M, respectively. In addition, the glucose biosensor system indicates excellent reproducibility (less than 5% R.S.D.) and good operational stability (over 2 weeks).     

SiO2/SnO2/Sb2O5 microporous ceramic material for immobilization of Meldola's blue: application as an electrochemical sensor for NADH

The mixed oxide SiO(2)/SnO(2), containing 25 wt% of SnO(2), determined by X-ray fluorescence, was prepared by the sol-gel method and the porous matrix obtained was then grafted with Sb (V), resulting the solid designated as (SiSnSb). XPS indicated 0.7% of Sb atoms on the surface. Sb grafted on the surface contains Br?nsted acid centers (SbOH groups) that can immobilize Meldola's blue (MB(+)) cationic dye onto the surface by an ion exchange reaction, resulting the solid designated as (SiSnSb/MB). In the present case a surface concentration of MB(+)=2.5×10(-11) mol cm(2) on the surface was obtained. A homogeneous mixture of the SiSnSb/MB with ultra pure graphite (99.99%) was pressed in disk format and used to fabricate a working electrode that displayed an excellent specific electrocatalytic response to NADH oxidation, with a formal potential of -0.05 V at pH 7.3. The electrochemical properties of the resulting electrode were investigated thoroughly with cyclic voltammetric and chronoamperometry techniques. The proposed sensor showed a good linear response range for NADH concentrations between 8×10(-5) and 9.0×10(-4) mol L(-1), with a detection limit of 1.5×10(-7) mol L(-1). The presence of dopamine and ascorbic acid did not show any interference in the detection of NADH on this modified electrode surface.     

Botanical micelle and its application for direct electrochemical biosensor

This paper is concerned about the entrapment of horseradish peroxidase (HRP) within botanical inositol hexakisphosphoric (IP(6)) micelles for the preparation of enzyme biosensor. The good affinity of IP(6) micelles with the enzyme provides naturally biocompatible microenvironment for the enzyme immobilization, achieving the direct electron transfer between HRP and electrode surface. The resulting biosensor to H(2)O(2) detection exhibits a low detection limit of 0.1 μmol L(-1) (S/N = 3), a quick response time (3s), and a long-term stability. The apparent Michaelis-Menten constant is quite tiny about 0.0016 mmol L(-1).     

Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.     

HRP/[Zn-Cr-ABTS] redox clay-based biosensor: design and optimization for cyanide detection

A novel inexpensive and simple amperometric biosensor, based on the immobilization of HRP into redox active [Zn-Cr-ABTS] layered double hydroxide, is applied to the determination of cyanide. The electrochemical transduction step corresponds to the reduction at 0.0 V of ABTS+* enzymatically formed in the presence of H2O2. The biosensor has a fast response to H2O2 (8s) with a linear range of 1.7 x 10(-9) to 2.1 x 10(-6) M and a sensitivity of 875 mA M(-1) cm(-2). The apparent Michaelis-Menten constant (KMapp) is 12 microM. The detection of cyanide is performed via its non competitive inhibiting action on the HRP/[Zn-Cr-ABTS] electrode. The concentration range of the linear response and the apparent inhibition constant (ki) are 5 x 10(-9) to 4 x 10(-8) and 1.4 x 10 (-7) M, respectively.     

Novel snowflake-like Pt-Pd bimetallic clusters on screen-printed gold nanofilm electrode for H2O2 and glucose sensing

Novel snowflake-like Pt-Pd bimetallic nanoclusters (Pt-PdBNC) were synthesized on a screen-printed gold nanofilm electrode (SPGFE) substrate by electrochemically reducing precursors with a new constant potential/multi-potential step deposition strategy. The electrocatalytic behavior of the modified electrode (SPGFE/Pt-PdBNC) towards H(2)O(2) was investigated. The results indicate that the as-prepared Pt-PdBNC significantly enhances the electrochemical reduction of H(2)O(2) in neutral media, exhibiting preferable electrocatalytic performance compared to Pt and Pd monometallic nanoclusters. Under optimum conditions, SPGFE/Pt-PdBNC offers linear responses for H(2)O(2) in the concentration range from 0.005 to 6 mM with an ultrahigh sensitivity of 804 mA M(-1) cm(-2) and excellent selectivity. Furthermore, glucose oxidase was immobilized on the Pt-PdBNC structure, and the fabricated biosensor presents favorable properties for glucose sensing.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

Bacterial sensors based on Acidithiobacillus ferrooxidans Part I. Fe2+ and S2O32- determination

An amperometric bacterial sensor with current response to Fe(2+) and S(2)O(3)(2-) ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O(2) flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe(2+) ions up to 2.5 mmol L(-1) with a detection limit of 9 x 10(-7) mol L(-1) and a sensitivity of 0.25 A L mol(-1). The response of the biosensor is 84 s for a determination of 2 x 10(-4) mol L(-1) Fe(2+). Optimizing the Fe(2+) determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 degrees C under 290 rpm solution stirring. S(2)O(3)(2-) concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L(-1). Sensitivity was 0.20 A L mol(-1) with a response time of 207 s for a 2 x 10(-4) mol L(-1) S(2)O(3)(2-) concentration.     

Nanographene-based tyrosinase biosensor for rapid detection of bisphenol A

Hydrophilic nanographene (NGP) prepared by ball milling of graphite was used as the support to construct a novel tyrosinase biosensor for determination of bisphenol A (BPA). The performances of the nanographene-based tyrosinase biosensor were systematically compared with those of multiwall carbon nanotubes (MWNTs) modified tyrosinase biosensors. The results indicated that the nanographene-based tyrosinase biosensor provided significant advantages over MWNTs-based tyrosinase biosensor in term of response, repeatability, background current and limit of detection (LOD), which could be attributed to its larger specific surface area and unique hierarchical tyrosinase-NGP nanostructures. The nanographene-based tyrosinase biosensor displayed superior analytical performance over a linear range from 100 nmol L(-1) to 2000 nmol L(-1), with LOD of 33 nmol L(-1) and sensitivity of 3108.4 mA cm(-2)M(-1). The biosensor was further used for detecting BPA (leaching from different vessels) in tap water, and the accuracy of the results was validated by high performance liquid chromatography (HPLC). The nanographene-based tyrosinase biosensor proved to be a promising and reliable tool for rapid detection of BPA leached from polycarbonate plastic products and for on-site rapid analysis of emergency pollution affairs of BPA.     

Bacterial sensors based on Acidithiobacillus ferrooxidans Part II. Cr(VI) determination

The aerobic acidophilic bacterium Acidithiobacillus ferrooxidans oxidizes Fe(2+) and S(2)O(3)(2-) ions by consuming oxygen. An amperometric biosensor was designed including an oxygen probe as transducer and a recognition element immobilized by a suitable home-made membrane. This biosensor was used for the indirect amperometric determination of Cr(2)O(7)(2-) ions owing to methods based on a mediator (Fe(2+)) or titration. Using the mediator, the biosensor response versus Cr(2)O(7)(2-) was linear up to 0.4 mmol L(-1), with a response time of, respectively, 51 s (2 x 10(-5) mol L(-1) Cr(2)O(7)(2-)) and 61 s (6 x 10(-5) mol L(-1) Cr(2)O(7)(2-)). The method sensitivity was 816 microA L mol(-1). Response time and measurement sensitivity depended on membrane material and technique for biomass immobilization. For example, their values were 90 s-200 microA L mol(-1) when using a glass-felt membrane and 540 s-4.95 microA L mol(-1) with a carbon felt one to determine a concentration of 2 x 10(-5) mol L(-1) Cr(2)O(7)(2-). For the titration method, the biosensor is used to determine the equivalence point. The relative error of quantitative analysis was lower than 5%.     

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.     

Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection

Polyoxometalates (H(3)PW(12)O(40), H(4)SiW(12)O(40) and H(3)PMo(12)O(40)) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H(2)O(2) to form a blue color in aqueous solution. Among them, H(3)PW(12)O(40) (PW(12)) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H(3)PW(12)O(40)/graphene exhibited higher activity than H(3)PW(12)O(40) in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H(2)O(2)/TMB system provides a simple, accurate approach to colorimetric detection for H(2)O(2) or glucose. The colorimetric method based on POMs showed good response toward H(2)O(2) and glucose detection with a linear range from 1.34×10(-7) to 6.7×10(-5) mol/L and 1×10(-7) to 1×10(-4) mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay.     

Multiwalled carbon nanotubes dispersed in carminic acid for the development of catalase based biosensor for selective amperometric determination of H(2)O(2) and iodate

We report the preparation of stable dispersion of multiwalled carbon nanotubes (MWCNTs) using carminic acid (CA) as a dispersing agent. The transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) results confirmed that MWCNT is well dispersed in CA aqueous solution and CA has been well adsorbed at MWCNT walls. Fourier transform infrared (FTIR) and UV-vis absorption spectra results also confirmed the adsorption of CA at MWCNT. To develop a highly selective amperometric biosensor for H(2)O(2) and iodate, the model enzyme catalase (CAT) was immobilized at CACNT modified glassy carbon electrode surface. The immobilized CAT exhibits well defined quasi reversible redox peaks at a formal potential (E°') of -0.559V in 0.05M pH 7 phosphate buffer solution (PBS). The proposed CAT/CACNT biosensor exhibits excellent amperometric response towards H(2)O(2) and iodate in the linear concentration range between 10μM to 3.2mM and 0.01-2.16mM. The sensitivity values are 287.98μAmM(-1)cm(-2) and 0.253mAmM(-1)cm(-2), respectively. Moreover, the developed CAT biosensor exhibits high affinity for H(2)O(2) and iodate with good selectivity.     

Mechanism of thyroxine-mediated oxidation of reduced nicotinamide adenine dinucleotide in peroxidase-H2O2 system.

The oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the horseradish peroxidase (HRP)-H2O2 system is greatly increased by the addition of thyroxine or related compounds. On the basis of a study of the rate of NADH oxidation in the presence of various concentrations of thyroxine, it is clear that thyroxine acts as a catalyst for NADH oxidation. Spectral changes of a HRP-H2O2 complex (compound I) indicate that thyroxine acts as an electron donor to both compounds I and II. The rate of electron donation from thyroxine is much faster than that from NADH. The HRP-H2O2 system requires 0.83 mol of O2 for the oxidation of 1 mol of NADH. Ferricytochrome c is reduced to ferrocytochrome c by the system, and causes an inhibition of O2 consumption which can be abolished by superoxide dismutase. JUDGING FROM THE INHIBITION OF O2 uptake by ferricytochrome c, about 54% of the total flux of electrons from NADH to oxygen appears to proceed by way of O2-. These results suggest that the initial step of thyroxine-mediated NADH oxidation by HRP and H2O2 is the formation of oxidized thyroxine, a phenoxy radical, which attacks NADH to produce NAD.     

HRP-based biosensor for monitoring rifampicin

Pyrrole was electropolymerized onto a Pt electrode in the presence of LiClO(4) and horseradish peroxidase (HRP). This HRP-based biosensor has been used for the amperometric detection of rifampicin (RIF) in the presence of a constant concentration of H(2)O(2). The C(H(2)O(2)) as well as the applied potential (E(ap)) and the pH of the phosphate buffer have simultaneously been optimized through a central composite design. Under these conditions, repeatability, reproducibility, and stability of the modified electrode have been analyzed. The detection limit for RIF has been calculated taking into account the probability of false-positive (alpha) and -negative (beta), reaching a value of 5.06x10(-6) mol dm(-3). The biosensor was applied to the determination of RIF in pharmaceutical preparations and biological samples.     

Nano polyurethane-assisted ultrasensitive biodetection of H(2)O(2) over immobilized microperoxidase-11

Nanostructured polyurethane (PU) synthesized by an emulsion polymerization with narrow size distribution was employed for the first time directly as a novel matrix for enzyme immobilization to develop sensitively amperometric biosensors. When Microperoxidase-11 (MP-11) was selected as a model protein, the resulting hydrogen peroxide (H(2)O(2)) biosensor exhibited improved sensitivity of 29.6μAmM(-1)cm(-2) with quite good response time of (1.3±0.4)s and remarkable limit of detection as low as 10pM (S/N 3) over existing protocols. A linear calibration curve for hydrogen peroxide was obtained up to 1.3μM under the optimized conditions with a relative low calculated Michaelis-Menten constant (K(M)(app)) (1.87±0.05)μM, which indicated the enhanced enzymatic affinity of MP-11 to H(2)O(2) via PU. The possible interferents had negligible effect on the response current and time of the prepared biosensor. Results suggest that the PU nanoparticles (PU-NPs) with good biocompatibility and sufficient interfacial adhesion hold promise as an attractive support material for construction of ultrasensitive amperometric biosensor.     

Optical detection of organophosphorus compounds based on Mn-doped ZnSe d-dot enzymatic catalytic sensor

In this paper, we report a sensitive and selective method for detection of organophosphorus compounds (OPs) based on Mn:ZnSe d-dots-enzyme-hydrogen peroxide (H(2)O(2)) fluorescence quenching system. Acetylcholine esterase (AChE) can hydrolyze acetylcholine (ACh) to choline. Subsequently, choline oxidase (ChOx) oxidizes choline to generate H(2)O(2). The enzyme-generated H(2)O(2) can quench the fluorescence of Mn:ZnSe d-dots. When paraoxon are introduced in solution, it can interact with the active centers of AChE and decrease the enzyme activity. This leads to the decrease of the H(2)O(2) production and then the fluorescence quenching rate of Mn:ZnSe d-dots. Experimental results showed that the enzyme inhibition percentage of Mn:ZnSe d-dots-ChOx-AChE-ACh system was proportional to the logarithm of paraoxon in the range 4.84×10(-11) to 4.84×10(-6) mol/L with the detection limit (S/N=3) of 1.31×10(-11) mol/L. The proposed biosensor has been employed for quick determination of paraoxon in tap water and milk samples with satisfactory reproducibility and accuracy. This nano-biosensor was proved to be sensitive, rapid, simple and tolerance of most interfering substances.