Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).     

Putrescine biosensor based on putrescine oxidase from Kocuria rosea

The novel putrescine oxidase based amperometric biosensor selectively measures putrescine, which can be considered as an indicator of microbial spoilage. Putrescine oxidase (PUOX, EC 1.4.3.10) was isolated from Kocuria rosea (Micrococcus rubens) by an improved and simplified purification process. Cells were grown on brain heart infusion medium supplemented with putrescine. Cell-free extract was prepared in Tris buffer (pH 8.0) by Bead-beater. A newly elaborated step based on three-phase partitioning (TPP) was applied in the purification protocol of PUOX. The purified enzyme was immobilized on the surface of a spectroscopic graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinking agent. This modified working electrode was used in wall-jet type amperometric cell together with the Ag/AgCl (0.1M KCl) reference electrode and a platinum wire as auxiliary electrode in flow injection analysis system (FIA). Hydrogel composition, pH and potential dependence were studied. Optimal working conditions were 0.45mLmin(-1) flow rate of phosphate buffer (66mM, pH 8.0) and +50mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25mM putrescine, while the detection limit was 5μM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.     

Layer-by-layer hydroxymethyl ferrocene modified sensor for one-step flow/stop-flow injection amperometric immunoassay of alpha-fetoprotein

A rapid one-step flow/stop-flow injection amperometric immunoassay for alpha-fetoprotein (AFP) using a novel home-produced electrochemical sensor was proposed. The sensor was prepared using layer-by-layer adsorption of positively charged poly(allylamine) (PAA) and negatively charged hydroxymethyl ferrocene on a screen-printed electrode (SPE). The electrochemistry of the immobilized ferrocene moieties showed a surface-controlled electrode process. Based on an electrochemical enzyme-linked immunoassay with the immobilized ferrocene moieties as an electron transfer mediator between the electrode and the horseradish peroxidase (HRP)-labeled anti-AFP antibody, a calibration curve with two linear ranges from 5 to 20 and 20 to 150 ng ml-1 and a detection limit of 2 ng ml-1 for AFP determination was obtained under the optimized conditions of 0.891 ml min-1 flow rate, 20 microl injection volume and +25 mV applied potential. The sensor showed good repeatability and reproducibility and retained more than 95% of its original signal after 15 days of storage. The proposed method eliminated the need for washing and addition of any substrate or mediator. The complete assay could be handled in less than 25 min with a one-step injection of a 40 microl sample solution. The proposed method would be valuable for the diagnosis and monitoring of carcinoma and its metastasis.     

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Graphite-Teflon composite bienzyme electrodes for the determination of L-lactate: application to food samples.

A bienzyme amperometric graphite-Teflon composite biosensor, in which lactate oxidase (LOD) and peroxidase, together with the mediator ferrocene, are incorporated into the electrode matrix, was developed for the determination of L-lactate in food samples such as wine and yogurt by using both batch- and flow-injection modes. This bienzyme electrode was fabricated by simple physical inclusion of the enzymes and the mediator in the bulk of the graphite-Teflon matrix. A Teflon content of 70%, an applied potential of 0.00 V, and a pH of 7.4 were employed as working conditions. The composite bioelectrode exhibited long-term operation because of the renewability of its surface by polishing. Reproducible amperometric responses were achieved with different electrodes fabricated from different composite matrices, and no significant loss of the enzyme activity occurred after 6 months of storage at 4 degrees C. Detection limits for L-lactate of 1.4 and 0.9 microM were obtained by batch amperometry in stirred solutions and flow-injection with amperometric detection, respectively. An interferences study with different substances which may be present in wine and yogurt together with L-lactic acid demonstrated very good selectivity for the determination of this analyte. The bienzyme composite electrode was applied to the determination of L-lactic acid in red wine and shaken yogurt, and the methods were validated by comparing these results with those obtained by applying a recommended reference method.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Chiral analysis of amino acids using electrochemical composite bienzyme biosensors.

The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.     

Voltammetric determination of epinephrine by White rot fungi (Phanerochaete chrysosporium ME446) cells based microbial biosensor

The lyophilized biomass of White rot fungi (Phanerochaete chrysosporium ME446) was immobilized in gelatine using glutaraldehyde crosslinking agent on a Pt working electrode. The fungal cells retained their laccase activity under entrapped state. The immobilized cells were used as a source of laccase to develop amperometric epinephrine biosensor. The catalytic action of the laccase in the biosensor released an epinephrinequinone as a result of redox activity, thereby causing an increase in the current. The optimal working conditions of the biosensor were carried out at pH 4.5 (50 mM acetate buffer containing 100 mM K(3)Fe(CN)(6)), and 20°C. The sensor response was linear over a range of 5-100 μM epinephrine. The detection limit of the biosensor was found to be 1.04 μM. In the optimization and characterization studies of the microbial biosensor some parameters such as effect of fungi and gelatine amount, percentage of glutaraldehyde on the biosensor response and substrate specificity were carried out. In the application studies of the biosensor, sensitive determination of epinephrine in pharmaceutical ampules was investigated.     

Development of a screen-printed amperometric biosensor for the determination of L-lactate dehydrogenase level.

We attempted to develop a screen-printed biosensor for the amperometric determination of L-lactate dehydrogenase (LDH) level on the basis of NAD(+)/NADH-dependent dehydrogenase reaction. The printing ink for the working electrode consisted of L-lactate, NAD(+), composite polymer of hydroxyethyl cellulose with ethylene glycol, 3,4-dihydroxybenzaldehyde (3,4-DHB) as an electron transferring mediator, and graphite as the conducting material. The 3,4-DHB was electropolymerized on the carboneous working electrode by potential cycling between -200 and +300 mV vs. Ag/AgCl reference electrode. Through the electrocatalytic reaction with immobilized 3,4-DHB, the NADH generated by the LDH reaction could be efficiently oxidized at lower potential than the unmodified carbon electrode. The analytical performance of the electrode was characterized in terms of linear sensing range and detection limit for LDH. The response from the developed biosensor was linear up to 500 U/l of LDH, and the detection limit of 50 U/l was observed at the signal-to-noise ratio of 3.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

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.     

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)₂Cl₂ as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm². The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.     

An amperometric biosensor for polyphenolic compounds in red wine

In the present work, a biosensor was developed with Laccase Coriolus Versicolor as the biological reconnaissance element immobilized on derivatized polyethersulphone membranes and applied to a Pt-Ag, AgCl US electrode base. Its application to several polyphenols usually found in red wine (caffeic acid, gallic acid, catechin, rutin, trans-resveratrol, quercetin and malvidin) was tested. It was observed that an amperometric response was obtained for catechin at +100 mV (versus Ag, AgCl) and caffeic acid at -50 mV in acetate buffer solutions (pH 4.5) having 12% ethanol. At pH 3.5 and +100 mV the biosensor was sensitive to both substrates and their response was additive. A limit of detection of 1.0 x 10(-6) M, linearity ranging from 2.0 to 14.0 x 10(-6) M, high sensitivity (0.0566 mAM(-1)) and reproducibility (R.S.D. <10%) were achieved for equimolar mixed solutions of catechin and caffeic acid. Under the same experimental conditions the other polyphenols tested individually did not yield any biosensor response. The application of the biosensor to red wine samples required a previous solid phase extraction for polyphenols enrichment. In fact, attempts to apply the biosensor in red wine using the "standard addition" methodology showed that large interferences occurred, as was to be expected. Reduction currents of -0.33 +/- 0.03 nA were obtained when the biosensor was used with the wine extract at +100 mV. This current could be ascribed to catechin and caffeic acid, although some interference by other polyphenols at the matrix level seemed to persist. The present biosensor showed promising applications for the wine analysis in future.     

Hydrogen peroxide detection at a horseradish peroxidase biosensor with a Au nanoparticle-dotted titanate nanotube|hydrophobic ionic liquid scaffold

In this work, a novel sensing scaffold, consisting Au nanoparticle (GNP)-dotted TiO(2) nanotubes (TNTs) as the rigid material and the hydrophobic ionic liquid (HIL), 1-decyl-3-methylimidazolium tetrafluoroborate, as the entrapping agent, was applied to facilitate the electron transfer of horseradish peroxidase (HRP) on a glassy carbon electrode. GNPs were immobilised on the TNTs in our work using a one-step reduction of HAuCl(4)·3H(2)O by sodium borohydride in the presence of sodium citrate as a stabilising reagent. The morphology and composition of the as-synthesised composite materials were characterised by transmission electron microscopy, scanning electron microscopy, X-ray diffraction and Fourier-transform infrared spectroscopy. Cyclic voltammetry of HRP at the modified electrode presented a pair of reproducible, quasi-reversible redox peaks with a peak-to-peak separation of 69 mV, indicating electron transfer between HRP and composite electrode. The GNP-TNT|HIL|HRP electrode was then applied to the detection of H(2)O(2) in a pH 7.0 phosphate buffer using chronoamperometry. The biosensor exhibited a linear response in the 15-750 μM range, and a limit of detection of 2.2 μM. The biosensor also exhibited stability with 90% of the detection signal retained over a two-week duration.     

Electrochemical study of a new methylene blue/silicon oxide nanocomposition mediator and its application for stable biosensor of hydrogen peroxide

A novel organic-inorganic nanocomposite of methylene blue (MB) and silicon oxide was synthesized and characterized by TEM, FTIR, and UV-vis. The as-prepared material was able to transfer the electron of the MB to electrode and was different from other SiO2 spheres structurally. It can be used as mediator to construct a biosensor with horseradish peroxidase (HRP) coimmobilized in the gelatine matrix and cross-linked with formaldehyde. The resulting biosensor exhibited fast amperometric response and good stability to hydrogen peroxide (H2O2). The linear range for H2O2 determination was from 1 x 10(-5) to 1.2 x 10(-3) M, with a detection limit of 4 x 10(-6) M based on S/N = 3. Moreover, the lifetime is more than 3 months under dry conditions at 4 degrees C.     

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.     

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.