Total phenol analysis of weakly supported water using a laccase-based microband biosensor

The monitoring of phenolic compounds in wastewaters in a simple manner is of great importance for environmental control. Here, a novel screen printed laccase-based microband array for in situ, total phenol estimation in wastewaters and for water quality monitoring without additional sample pre-treatment is presented. Numerical simulations using the finite element method were utilized for the characterization of micro-scale graphite electrodes. Anodization followed by covalent modification was used for the electrode functionalization with laccase. The functionalization efficiency and the electrochemical performance in direct and catechol-mediated oxygen reduction were studied at the microband laccase electrodes and compared with macro-scale electrode structures. The reduction of the dimensions of the enzyme biosensor, when used under optimized conditions, led to a significant improvement in its analytical characteristics. The elaborated microsensor showed fast responses towards catechol additions to tap water – a weakly supported medium – characterized by a linear range from 0.2 to 10 μM, a sensitivity of 1.35 ± 0.4 A M⁻¹ cm⁻² and a dynamic range up to 43 μM. This enhanced laccase-based microsensor was used for water quality monitoring and its performance for total phenol analysis of wastewater samples from different stages of the cleaning process was compared to a standard method.     

Modelling synergistic action of laccase-based biosensor utilizing simultaneous substrates conversion

The response of a laccase-based amperometric biosensor that acts in a synergistic manner was modelled digitally. A mathematical model of the biosensor is based on a system of non-linear reaction diffusion equations. The modelling biosensor comprises three compartments, an enzyme layer, a dialysis membrane and an outer diffusion layer. By changing input parameters the biosensor action was analysed with a special emphasis to the influence of the species concentrations on the synergy of the simultaneous substrates conversion. The digital simulation was carried out using the finite difference technique.     

A flow injection biosensor system for highly sensitive detection of 2,4,6-trichlorophenol based on preoxidation by ceric sulfate.

A flow injection biosensor system was proposed for the highly sensitive detection of 2,4,6-trichlorophenol (2,4,6-TCP). The system is based on the preoxidation by ceric sulfate to the corresponding benzoquinone (2,6-dichloro-1,4-benzoquinone: 2,6-DC-1,4-BQ), which was characterized using cyclic voltammetry, hydrodynamic voltammetry, and UV-vis spectrophotometry. The laccase-based biosensor used in this analytical system responded sensitively to 2,4,6-TCP after the preoxidation by ceric sulfate. The response could be based on the bioelectrocatalytic recycling of oxidation product (2,6-DC-1,4-BQ) between laccase membrane and the electrode, because the oxidation product (2,6-DC-1,4-BQ) of 2,4,6-TCP was an electrochemically reversible redox species. The signal current was linearly related to the 2,4,6-TCP concentrations in a dynamic range of 2 nM - 2 microM; the slope and the y-intercept of the straight line were 1150 nA microM(-1) and 0.88 nA, respectively. The detection limit was 1.2 nM (S/N = 3) for a 20 microl injection. Among a variety of chlorophenols and some phenolic compounds, the only interferent was 2,4-dichlorophenol.     

In-field monitoring of cleaning efficiency in waste water treatment plants using two phenol-sensitive biosensors

Two amperometric biosensors based on the enzymes cellobiose dehydrogenase (CDH) and quinoprotein-dependent glucose dehydrogenase (GDH), have been applied for monitoring the phenolic content in water samples, collected at different stages of a waste water treatment process, thus representing different cleaning levels of two waste water treatment plants (WWTPs). The biosensor measurements were performed in-field, compared with the results obtained by liquid chromatography-mass spectrometry and were further correlated with the cleaning efficiencies of the WWTPs. The effect of several potentially interfering compounds on the sensor response was also studied.The general purpose of the study was to evaluate the potential use of biosensors, not as quantitative tools for phenol analysis, but rather as screening tools indicating a certain trend, i.e. compounds present or not present, and potential correlation with sample toxicity. It was found that the biosensors and LC-MS results were not quantitatively comparable, however, both sensors could follow the decrease of the phenol content from the influent, primary treated and effluent waters. In addition, the correlation between biosensor inhibition and sample toxicity is discussed.     

Laccase Biosensor Based on N‐Succinimidyl‐3‐Thiopropionate‐Functionalized Gold Electrodes

A laccase biosensor, in which the enzyme was immobilized on N‐succinimidyl‐3‐thiopropionate (NSTP)‐modified gold electrodes, is reported. Two different approaches for the preparation of N‐succinimidyl‐terminated monolayers were evaluated: a) activation of a preformed 3‐mercaptopropionic acid (MPA) SAM by reaction with 1‐(3‐dimethylaminopropyl)‐ 3‐ethylcarbodiimide (EDC) and N‐hydroxysulfosuccinimide (NHS); b) assembling of dithiobisuccinimidyl propionate (DTSP). NSTP‐modified electrodes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Biosensors prepared by covalent binding of the enzyme and by cross‐linking with glutaraldehyde atop NSTP‐modified electrodes were compared in terms of sensitivity and operational range for caffeic acid. A much better analytical performance was found using the latter approach. Variables affecting the amperometric detection (enzyme loading, pH and applied potential) were optimized. The operational stability and characteristics of functioning of the laccase biosensor in terms of repeatability of the amperometric measurements, reproducibility with different biosensors and useful lifetime, were evaluated. The kinetic parameters of the enzyme reactions and the analytical characteristics of the corresponding calibration plots were calculated for eight phenolic compounds. Limits of detection of 0.07 μM, 0.05 μM and 0.09 μM were obtained for caffeic acid, catechol and 3,4‐dihydroxyphenylacetic acid (DOPAC), respectively. The practical usefulness of the developed biosensor was evaluated by estimating the “pool” of phenolic compounds in olive oil mill wastewaters (OMW).     

Characterization of graphite electrodes modified with laccase from Trametes versicolor and their use for bioelectrochemical monitoring of phenolic compounds in flow injection analysis

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 μM, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min⁻¹ and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP).     

Monitoring of environmental phenolic endocrine disrupting compounds in treatment effluents and river waters, Korea

The last two decades have witnessed growing scientific and public concerns over endocrine disrupting compounds (EDCs) that have the potential to alter the normal structure or functions of the endocrine system in wildlife and humans. In this study, the phenolic EDCs such as alkylphenol, chlorinated phenol and bisphenol A were considered. They are commonly found in wastewater discharges and in sewage treatment plant. In order to monitor the levels and seasonal variations of phenolic EDCs in various aquatic environments, a total of 15 water samples from the discharged effluent from sewage and wastewater treatment plants and river water were collected for 3 years. Ten environmental phenolic EDCs were determined by GC-MS and laser-induced fluorescence (LIF). GC-MS analysis revealed that most abundant phenolic EDCs were 4-n-heptylphenol, followed by nonlyphenol and bisphenol A during 2002-2003, while 4-t-butylphenol and 4-t-octylphenol were newly detected in aquatic environments in 2004.The category of phenolic EDCs showed similar fluorescence spectra and nearly equal fluorescence decay time. This makes it hard to distinguish each phenolic EDC from the EDCs mixture by LIF. Therefore, the results obtained from LIF analysis were expressed in terms of the fluorescence intensity of the total phenolic EDCs rather than that of the individual EDC. However, LIF monitoring and GC-MS analysis showed consistent result in that the river water samples had lower phenolic EDCs concentration compared to the effluent sample. This revealed a lower fluorescence intensity and the phenolic EDCs concentration in summer was lower than that in winter. For the validation of LIF monitoring for the phenolic EDCs, the correlation between EDCs concentration acquired from GC-MS and fluorescence intensity from LIF was obtained (R = 0.7379). This study supports the feasibility of the application of LIF into EDCs monitoring in aquatic systems.     

Simultaneous Determination of Epinephrine and Dopamine by Using Candida tropicalis Yeast Cells Immobilized in a Carbon Paste Electrode Modified with Single Wall Carbon Nanotube

A new electrochemical microbial biosensor system based on Candida tropicalis was developed for the fast detecting of dopamine and epinephrine. Candida tropicalis was immobilized in a carbon paste electrode (CPE) with single wall carbon nanotube (SWCNT). Immobilized cells were used as a origin of the polyphenol oxidase (PPO) to develop voltammetric epinephrine and dopamine biosensor. Voltammetric determination of phenolic compounds such as epinephrine and dopamine a simple technique which is available. Direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. Another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidizes the phenolic compounds into their related quinones. By this way, phenolic compounds are epinephrine and dopamine which were used in this study as well detected at different potentials. In this study differential pulse voltammetry and amperometry techniques were used for the determination of dopamine and epinephrine. The effect of varying the amounts of SWCNT and the response of microorganism to epinephrine was investigated to find the optimum composition of the sensor. The effects of pH and temperature were also examined. Increases in biosensor responses obtained by amperometric measurements were linearly related to dopamine concentrations between 0.025 and 0.25 mM and epinephrine concentrations between 0.01 and 0.1 mM. Limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.008 and 0.0023 mM, respectively. Finally, proposed system was applied to epinephrine and dopamine analysis in pharmaceutical drugs and synthetic serum and the results were compared with LC MS MS method.     

Quinoprotein glucose dehydrogenase modified carbon paste electrode for the detection of phenolic compounds

The development of an amperometric biosensor for the determination of phenolic compounds is described, using quinoprotein glucose dehydrogenase. The enzyme is integrated into carbon paste and its ability to donate electrons to oxidized phenolic compounds during glucose oxidation is exploited. The sensor response is based on electrochemical oxidation of the phenolic compound followed by its enzymatic regeneration when the bulk solution contains glucose and the electrode is potentiostated at +500 mV (vs. Ag/AgCl/0.1 mol/L KCl). As the result of the catalytic analyte regeneration the electrodes offer very sensitive measurements of redox species like p-aminophenol and hydroquinone and catecholamines such as epinephrine, norepinephrine, and dopamine. The sensor performance is characterized for the different substrates. Highest sensitivity is achieved for p-aminophenol which could be determined at sub-nanomolar level.     

Electrochemical sensor with flavin-containing monooxygenase for triethylamine solution

A bioelectronic sensor for triethylamine (TEA) was developed with a flavin-containing monooxygenase type 3 (FMO-3). The TEA biosensor consisted of a Clark-type dissolved-oxygen electrode and an FMO-3 immobilized membrane. The FMO-3 solution was mixed with a poly(vinyl alcohol) containing stilbazolium groups (PVA-SbQ), coated on to the dialysis membrane, and the membrane was irradiated with a fluorescent light to immobilize the enzyme. In order to amplify the biosensor output, a substrate regeneration cycle, obtained by coupling the monooxygenase with l-ascorbic acid (AsA) as reducing reagent system, was applied. The effect of pH on the determination of TEA was studied. The maximum response was achieved at pH >9.0. A drop of the phosphate buffer solution with the AsA was put on the sensing area of the oxygen electrode, and the FMO-3 immobilized membrane was placed on the oxygen electrode and covered with a supporting Nylon mesh net which was secured with a silicone O-ring. A measurement system for TEA solution was constructed using the FMO-3 biosensor, a personal computer, a computer-controlled potentiostat, and an A/D converter. The FMO-3 biosensor was used to measure TEA solution from 0.5 to 4.0 mmol L⁻¹ with 10.0 mmol L⁻¹ AsA. The biosensor also had good reproducibility, for example a 6.31% coefficient of variation for five measurements, and the output current was maintained over a few hours. In order to improve the selectivity of the TEA biosensor, three type of biosensor with FMO isomer types 1, 3, and 5 were constructed and used to measure nitrogen and sulfur compounds. The outputs of the isomer biosensors indicated individual patterns for each sample solution. The selectivity of TEA biosensor would be improved, and determination of sulfur and nitrogen compounds would be possible, by using the different output of biosensors prepared from different FMO isomers.     

Electrochemical detection of phenolic compounds using composite film of multiwall carbon nanotube/surfactant/tyrosinase on a carbon paste electrode

A new tyrosinase-based biosensor was developed for detection of phenolic compounds using composite film of multiwall carbon nanotube (MWCNT)/dimethylditetradecylammonium bromide (DTDAB)/tyrosinase (Tyr) on a Nafion-incorporated carbon paste electrode. The biosensor showed a sensitive electrochemical response to the reduction of the oxidation products of different phenolic compounds (phenol, catechol, p-cresol, and p-chlorophenol) by dissolved O₂ in the presence of the immobilized enzyme. The effects of pH, operating potential, MWCNT concentration, and the DTDAB/Tyr ratio on electrochemical response were explored for optimum analytical performance. The biosensor exhibited a linear response range of 1.5–25.0, 2.0–15.0, 2.0–15.0, and 2.5–25.0 μM and sensitivity of 2,900, 3,100, 3,100, and 1,500 μA/mM for phenol, catechol, p-cresol, p-chlorophenol, respectively. In addition, the response of the enzyme electrode showed Michaelis–Menten behavior at concentrations of the phenolic compounds higher than 5.0 μM. The stability and the application of the biosensor were also evaluated.     

Chitosan-based tyrosinase optical phenol biosensor employing hybrid nafion/sol-gel silicate for MBTH immobilization

The development of an optical biosensor based on immobilization of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in hybrid nafion/sol-gel silicate film and tyrosinase in chitosan film for the detection of phenolic compounds has been described. Tyrosinase was immobilized in chitosan film deposited on the hybrid nafion/sol-gel silicate film containing MBTH. The enzymatic oxidation product of phenolic compounds were stabilized through formation of adduct with MBTH to produce a maroon color adduct. The color intensity of adduct was found to increase proportionally with the increase of the substrate concentrations after 5 min exposure. The linearity of the biosensor towards phenol, catechol and m-cresol were in the respective concentration range of 0.5-7.0, 0.5-10.0 and 1.0-13.0 mg/L with detection limit of 0.18, 0.23 and 0.43 mg/L, respectively. The biosensor shows a good stability for at least 3 months.     

Encapsulation of tyrosinase within liposome bioreactors for developing an amperometric phenolic compounds biosensor

A novel tyrosinase (Tyr) biosensor based on liposome bioreactor and chitosan (CS) nano-composite has been developed for the detection of phenolic compounds. Liposome-based bioreactors were prepared by encapsulating the enzyme Tyr in l-α-phosphatidylcholine liposome resulting in spherical bioreactor with a mean diameter of 8.5?±?1.25 μm. The encapsulation efficiency and drug loading content of the Tyr-loaded liposome-based bioreactors were about 46.35?±?0.85 and 41.15?±?0.95 %, respectively. Porins were embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The glassy carbon electrode (GCE) was alternately immersed in CS and Tyr liposome bioreactor (TLB) to assemble bilayer films [(CS/TLB)/GCE]. The presence of Tyr in the biosensor was confirmed by scanning electron microscopy, cyclic voltammetry, and electrochemical measurements. The results indicated that the biosensor was applied to detect phenol with a broad linear range from 0.25 nM to 25 μM, the detection limit was brought down to 0.091 nM. The apparent Michaelis–Menten constant, K _m, for the enzymatic reaction was 34.78 μM. The novel biosensor exhibits good repeatability and stability. Such new biosensor based on encapsulation of Tyr within liposome bioreactors 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.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Comparison of different sample preparation treatments for the analysis of wine phenolic compounds in human plasma by reversed phase high-performance liquid chromatography

Phenolic compounds are common constituents of wine. Due to their healthy properties the analysis in human fluids is interesting within bioavailability evaluation. They have been reported not to be stable in human plasma, particularly at room temperature. Most sample treatments have been reported for a single compound. Our aim in this paper is to study sample handling control conditions and improve phenolic stability in human plasma samples. We tested various sample treatments to determine whether they could be used for analysing a set of phenolic compounds usually present in wines.The compounds studied were six phenolic acids, five flavonoids, trans-resveratrol and tyrosol. The effect of the following factors was explored: temperature, pH, the addition of antioxidants and the addition of anticoagulants.The results suggest that the plasma samples should be kept at temperatures below −20 °C before analysis and that 1% ascorbic acid plus 10 μl/ml o-phosphoric acid should be added. Anticoagulants (heparin or EDTA) do not play a significant role in the stability of polyphenolic compounds.The recovery values of a number of sample treatments (solid phase extraction, extraction with methanol, deproteinization, inhibition of enzymatic plasma activity) were compared. The recovery values for most phenolic compounds were better if the enzymatic plasma activity was inhibited and acidified ethanol was used for deproteinization.     

Composite Multienzyme Amperometric Biosensors for an Improved Detection of Phenolic Compounds

A biosensor design, in which glucose oxidase and peroxidase are coimmobilized by simple physical inclusion into the bulk of graphite‐Teflon pellets, is reported for the detection of phenolic compounds. This design allows the “in situ” generation of the H₂O₂ needed for the enzyme reaction with the phenolic compounds, which avoids several problems detected in the performance of single peroxidase biosensors as a consequence of the presence of a high H₂O₂ concentration. So, a much lower surface fouling was found at the GOD‐HRP biosensor in comparison with a graphite‐Teflon‐HRP electrode, suggesting that the controlled generation of H₂O₂ makes more difficult the formation of polymers from the enzyme reaction products. The construction of trienzyme biosensors, in which GOD, HRP and tyrosinase were coimmobilized into the graphite‐Teflon matrix is also reported, and their performance was compared with that of GOD‐HRP bienzyme electrodes. The practical applicability of the composite multienzyme amperometric biosensors was evaluated by the estimation of the phenolic compounds content in waste waters from a refinery, and the results were compared with those obtained by using a colorimetric official method based on the reaction with 4‐aminoantipyrine.     

Development of a tyrosinase biosensor based on gold nanoparticles-modified glassy carbon electrodes: Application to the measurement of a bioelectrochemical polyphenols index in wines

The preparation of a tyrosinase biosensor based on the immobilization of the enzyme onto a glassy carbon electrode modified with electrodeposited gold nanoparticles (Tyr-nAu-GCE) is reported. The enzyme immobilized by cross-linking with glutaraldehyde retains a high bioactivity on this electrode material. Under the optimized working variables (a Au electrodeposition potential of −200 mV for 60 s, an enzyme loading of 457 U, a detection potential of −0.10 V and a 0.1 mol l⁻¹ phosphate buffer solution of pH 7.4 as working medium) the biosensor exhibited a rapid response to the changes in the substrate concentration for all the phenolic compounds tested: phenol, catechol, caffeic acid, chlorogenic acid, gallic acid and protocatechualdehyde. A R.S.D. of 3.6% (n = 6) was obtained from the slope values of successive calibration plots for catechol with the same Tyr-nAu-GCE with no need to apply a cleaning procedure to the biosensor. The useful lifetime of one single biosensor was of at least 18 days, and a R.S.D. of 4.8% was obtained for the slope values of catechol calibration plots obtained with five different biosensors. The kinetic constants and the analytical characteristics were calculated for all the phenolic compounds tested. The Tyr-nAu-GCE was applied for the estimation of the phenolic compounds content in red and white wines. A good correlation of the results (r = 0.990) was found when they were plotted versus those obtained by using the spectrophotometric method involving the Folin-Ciocalteau reagent.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Flow-Cell Fibre-Optic Enzyme Sensor for Phenols

Abstract

A solid-state fibre-optic luminescent oxygen sensor was used for flow-through measurements. It acts as a transducer in a new flow-cell enzyme sensor arrangement. This arrangement comprises a flow path, sample injector, microcolumn with the immobilized enzyme, oxygen membrane and fibre-optic connector joined together to form an integral unit. Laccase enzyme was used as a recognition system which provided specific oxidation of the substrates with the dissolved oxygen being monitored. The assay procedure was optimized and performance of the new system studied. The sensor was applied to the determination polyphenol content in tea, brandy, etc. (quality control test). The sensitivity to some important phenolic compounds was tested with the view of industrial wastewater control applications.     

A novel tyrosinase biosensor based on hydroxyapatite-chitosan nanocomposite for the detection of phenolic compounds

A novel tyrosinase biosensor based on hydroxyapatite nanoparticles (nano-HA)-chitosan nanocomposite has been developed for the detection of phenolic compounds. The uniform and size controlled nano-HA was synthesized by hydrothermal method, and its morphological characterization was examined by transmission electron microscope (TEM). Tyrosinase was then immobilized on a nano-HA-chitosan nanocomposite-modified gold electrode. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the sensing film. The prepared biosensor was applied to determine phenolic compounds by monitoring the reduction signal of the biocatalytically produced quinone species at −0.2 V (vs. saturated calomel electrode). The effects of the pH, temperature and applied potential on the biosensor performance were investigated, and experimental conditions were optimized. The biosensor exhibited a linear response to catechol over a wide concentration range from 10 nM to 7 μM, with a high sensitivity of 2.11 × 10³ μA mM⁻¹ cm⁻², and a limit of detection down to 5 nM (based on S/N = 3). The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 3.16, 1.31 and 3.52 μM for catechol, phenol and m-cresol, respectively. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.