Stacked films immobilization of MBTH in nafion/sol-gel silicate and horseradish peroxidase in chitosan for the determination of phenolic compounds

The stacked-film immobilization of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in hybrid nafion/sol-gel silicate film and horseradish peroxidase (HRP) in chitosan, performed in order to allow the determination of phenolic compounds, was investigated via an optical method. The stacked films were deposited onto a microscope glass slide by a spin-coating technique. The quinone or free radical product formed by the enzymatic reactions of phenolic compounds interacts with MBTH to form azo-dye products, which can be measured spectrophotometrically at a wavelength of 500 nm. The color intensity of the product was found to increase in proportion to the phenolic concentration after 5 min of exposure. The response of the biosensor was linear over concentration ranges of 0.025–0.500, 0.010–0.070 and 0.050–0.300 mM for guaiacol, resorcinol and o-cresol, respectively, and gave detection limits of 0.010, 0.005 and 0.012 mM. The sensor exhibited good sensitivity and stability for at least two months.     

Use of lignins as components of background electrolyte in capillary electrophoresis

The electrophoretic behavior of two lignins of different compositions, i.e., spruce dioxane lignin and lignosulfonate, is studied. The lignins are shown to affect the electrophoretic behavior of negatively charged analytes, such as carboxylic acids and phenols; their migration time increases. The addition of lignins improves the analytical parameters of phenol quantification by capillary electrophoresis. By means of a simple non-modified capillary, a mixture of six phenols was separated (simple phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, and perchlorophenol) with the high resolution (up to 20) and efficiency [(1–5) × 10⁵ TPM]. The separation of six phenols takes 10 min, the lower limit of the analytical range makes 1 μg/mL, the relative standard deviation does not exceed 3%. The potency for the determination of simple phenol and m-cresol is shown on an example of the Verrukatsid medication within 7 min.     

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.     

Optimisation and validation of an automated solid phase extraction technique coupled on-line to enzyme-based biosensor detection for the determination of phenolic compounds in surface water samples

Summary A fully integrated screening system for phenolic compounds was developed incorporating on-line solid phase extraction, fractionation and biosensor detection. Two different types of biosensors, solid graphite and carbon paste electrodes incorporating the enzyme tyrosinase, were compared and used in the screening system. Interfacing of the solid phase extraction and fractionation with the biosensor detection was given special attention since the biosensors were not compatible with the organic modifier used for desorption of phenols from the solid phase extraction step. The system was validated with conventional analytical techniques. Surface water samples from the Ebro river were spiked with 1,10, and 25g L^–1 of catechol, phenol,p-cresol, respectively. Three out of seven samples were spiked and the correct samples were identified, containing phenols equivalent to the spiked concentrations.     

Amperometric biosensor based on tyrosinase immobilized in hydrotalcite-like compounds film for the determination of polyphenols

A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg–Al–CO₃ hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of a positively charged HTLc/GCE by electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by a direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature, and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave a linear response range of 3–300, 0.888–444, and 0.066–396 μM with a detection limit (S/N = 3) of 0.1, 0.05, and 0.003 μM for catechol, caffeic acid, and quercetin, respectively. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.     

Highly sensitive choline biosensor based on carbon nanotube-modified Pt electrode combined with sol-gel immobilization

A novel amperometric choline biosensor has been fabricated with choline oxidase (ChOx) immobilized by the sol-gel method on the surface of multi-walled carbon nanotubes (MWCNT) modified platinum electrode to improve the sensitivity and the anti-interferential property of the sensor. By analyzing the electrocatalytic activity of the modified electrode by MWCNT, it was found that MWCNT could not only improve the current response to H₂O₂ but also decrease the electrocatalytic potential. The effects of experimental variables such as the buffer solutions, pH and the amount of loading enzyme were investigated for the optimum analytical performance. This sensor shows sensitive determination of choline with a linear range from 5.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol/L when the operating pH and potential are 7.2 and 0.15 V, respectively. The detection limit of choline was 5.0 × 10⁻⁷ mol/L. Selectivity for choline was 9.48 μA·(mmol/L)⁻¹. The biosensor exhibits excellent anti-interferential property and good stability, retaining 85% of its original current value even after a month. It has been applied to the determination of choline in human serum. Translated from Chinese Journal of Analytical Chemistry, 2006, 34(7): 910–914 (in Chinese)     

Ultrasound-assisted derivatization of phenolic compounds in spiked water samples before pervaporation, gas chromatographic separation, and flame lonization detection

Summary A fully automated method, based on continuous ultrasound-assisted derivatization coupled with pervaporation before gas chromatographic separation and flame ionization detection, has been developed for the determination of phenol and cresols in water. Spiked water samples were doped with acetic anhydride and dipotassium hydrogen phosphate, before introduction into the flow system, to achieve catalytic acetylation of the target compounds. A multivariate study was performed to optimize the main factors affecting the derivatization process. The correlation coefficients, r, of the calibration plots obtained were better than 0.999 for cresols and better than 0.99 for phenol. Detection limits were 0.02 μg mL¹ for phenol, o-cresol, andp-cresol, and 0.05 μg mL¹ form-cresol. Reproducibility and repeatability, expressed as relative standard deviation, ranged from 2.0–3.9% and from 1.0–3.5% respectively.     

Determination of Phenolic Compounds Based on Co‐Immobilization of Methylene Blue and HRP on Multi‐Wall Carbon Nanotubes

This work evaluated an amperometric biosensor based on multi‐wall carbon nanotubes (MWCNT), chemically modified with methylene blue (Met) and horseradish peroxidase (HRP), for detection of phenolic compounds. The dependences of the biosensor response due to the enzyme immobilization procedure, HRP amounts, pH and working potential were investigated. The amperometric response for catechol using the proposed biosensor showed a very wide linear response range (1 to 150 μmol L^?1), good sensitivity (50 nA cm^?2 μmol^?1 L), excellent operational stability (after 300 determinations the response remained at 97%) and very good storage stability (lifetime>3 months). Based on all these characteristics, it is possible to affirm that the material is promising for phenol detection due to its good electrochemical response and enzyme stabilization. The biosensor response for various phenolic compounds was investigated.     

Tyrosinase/laccase bienzyme biosensor for amperometric determination of phenolic compounds

Tyrosinase/laccase bienzyme biosensor for amperometric determination of phenolic compounds was constructed. Enzymes were immobilized in titania gel matrix. The obtained biosensor was successfully used for determination of 2,6-dimethoxyphenol, 4-tertbutylcatechol, 4-methylcatechol, 3-chlorophenol and catechol. The highest sensitivity and the widest linear range were noticed for catechol, 234 mA L mol^− 1 and 2.0 × 10^− 7–3.2 × 10^− 5 mol/L, respectively. Detection limit for catechol, at signal-to-noise ratio of 3 was 1.3 × 10^− 7 mol/L.     

Acute Toxicity of Cresols to Pseudomonas — An Initial Oxygen Uptake Method

Acute toxicity of cresols to both Pseudomonas I and II was estimated by an initial oxygen uptake method. Inhibition studies of toluene and cresols on the oxidation of either benzoate by Pseudomnas I or phenol by Pseudomonas II were analyzed and expressed as oxygen uptake rates. Double reciprocal plots for the inhibiton by cresols of oxygen uptake in Pseudomonas, two physical constants, Vmaxi and Ki, were obtained. The Vmaxi of o?, m? and p-cresol were 80%, 81% and 57% of Vmax in Pseudomnas I, and 10%, 25% and 36% in Pseudomonas II, respectively. Thus, the toxicity to Pseudomonas I decreases in the order p- > o- ≥ m-cresol, whereas to Pseudomonas II, the order is changed to o- > m- > p-cresol. This difference in the toxicity order is probably due to the allosteric effect of p-cresol towards Pseudomonas II. Inasmuch as most compounds inhibit noncompetively, the relative toxicity of different compounds can be estimated by a new toxicity parameter RI (relative inhibition) which is defined as 100/Ki. By comparing the RI value of each compound, the toxicity to Pseudomonas I decreases in the order m-chlorophenol > p-cresol > p-chlorophenol > o-cresol ≥ m-cresol > o-chlorophenol > toluene > phenol.     

Solid phase extraction and uptake properties of multi-walled carbon nanotubes of different dimensions towards some nitro-phenols and chloro-phenols from water

Equilibrium sorption studies and solid phase extraction (SPE) of various phenols (Phenol (Ph), 2-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP)) on oxidized and raw multi-walled carbon nanotubes (MWCNTs) of various external diameters (10–20, 10–30, 20–40, 40–60 and 60–100?nm) and various lengths (short: 1–2?µm and long: 5–15?µm) were tested. Equilibrium sorption studies showed that 2-NP fits the Langmuir isotherm model (LIM), while the other phenolic compounds fit the Freundlich isotherm model (FIM). There was generally an inverse relation between external diameter of MWCNT and its sorption capacity towards phenolic compounds. Long MWCNT showed higher sorption capacity than short MWCNT. Thus dimensions of MWCNT play a role in retaining the sorbed molecules. Oxidation of MWCNT caused a decrease in sorption capacity of phenolic compounds of lower acidity where hydrophobic interaction is predominant, while it caused an increase in sorption capacity of phenolic compounds of higher acidity where H-bonding is predominant. The dependence of sorption on the acidity of phenols (pK_a values) indicates that the basic groups (pyrone-like groups) on the MWCNT surface play a role in the sorption process. In SPE experiments, it was found that hydrogen peroxide-oxidized MWCNT of external diameter 40–60?nm and length 5–15?µm was the best extractant at pH 6.5 using acetonitrile as eluting solvent. The optimized SPE procedure gave detection limits range: 0.027–0.202?ng?mL^?1 within the studied concentration range (10–100?ng?mL^?1). Application of the optimum SPE method on spiked tap water, reservoir water and stream water gave recovery range of 84.3–100% for 2-CP, 3-CP and 4-CP (%RSD range 2.8–9.2%); while Ph, 2-NP, 4-NP, and 2,4-DNP gave recoveries <67.6%.     

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at −0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.     

A new bacterial biosensor for trichloroethylene detection based on a three-dimensional carbon nanotubes bioarchitecture

Trichloroethylene (TCE), a suspected human carcinogen, is one of the most common volatile groundwater contaminants. Many different methodologies have already been developed for the determination of TCE and its degradation products in water, but most of them are costly, time-consuming and require well-trained operators. In this work, a fast, sensitive and miniaturised whole cell conductometric biosensor was developed for the determination of trichloroethylene. The biosensor assembly was prepared by immobilising Pseudomonas putida F1 bacteria (PpF1) at the surface of gold interdigitated microelectrodes through a three-dimensional alkanethiol self-assembly monolayer/carbon nanotube architecture functionalised with Pseudomonas antibodies. The biosensor response was linear from 0.07 to 100 μM of TCE (9–13,100 μg L⁻¹). No significant loss of the enzymatic activity was observed after 5 weeks of storage at 4 °C in the M457 pH 7 defined medium (two or three measurements per week). Ninety-two per cent of the initial signal still remained after 7 weeks. The biosensor response to TCE was not significantly affected by cis-1,2-dichloroethylene and vinyl chloride and, in a limited way, by phenol. Toluene was the major interference found. The bacterial biosensor was successfully applied to the determination of TCE in spiked groundwater samples and in six water samples collected in an urban industrial site contaminated with TCE. Gas chromatography–mass spectrometric analysis of these samples confirmed the biosensor measurements.     

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.     

The Construction of Glucose Biosensor Based on Platinum Nanoclusters—Multiwalled Carbon Nanotubes Nanocomposites

One-step synthesis method was proposed to obtain the nanocomposites of platinum nanoclusters and multiwalled carbon nanotubes (PtNCs–MWNTs), which were used as a novel immobilization matrix for the enzyme to fabricate glucose biosensor. The fabrication process of the biosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, atomic force microscopy and scanning electron microscope. Due to the favorable characteristic of PtNCs–MWNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0 μM–12.1 mM), low detection limit (1.0 μM), high sensitivity (12.8 μA mM⁻¹), rapid response time (within 6 s). The apparent Michaelis–Menten constant ( K_m^\textapp K_m^{\text{app}} ) is 2.1 mM. The performance of the resulting biosensor is more prominent than that of most of the reported glucose biosensors. Furthermore, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples.     

Multivariate data analysis of dynamic amperometric biosensor responses from binary analyte mixtures-application of sensitivity correction algorithms

In this paper, it is demonstrated that a single-receptor biosensor can be used to quantitatively determine each analyte in binary mixtures using multivariate data analysis tools based on the dynamic responses received from flow injection peaks. Mixtures with different concentrations of two phenolic compounds, catechol and 4-chlorophenol, were measured with a graphite electrode modified with tyrosinase enzyme at an applied potential of −50 mV versus Ag/AgCl. A correction algorithm based on measurements of references in-between samples was applied to compensate for biosensor ageing as well as differences caused by deviations between biosensor preparations. After correction, the relative prediction errors with partial least squares regression (PLS-R) for catechol and 4-chlorophenol were 7.4 and 5.5%, respectively, using an analysis sequence measured on one biosensor. Additional validation mixtures of the two phenols were measured with a new biosensor, prepared with the same procedure but with a different batch of tyrosinase enzyme. Using the mixture responses for the first sensor as a calibration set in PLS-R, the relative prediction errors of the validation mixtures, after applying correction procedures, were 7.0% for catechol and 16.0% for 4-chlorophenol. These preliminary results indicate that by applying correction algorithms it could be possible to use less stable biosensors in continuous on-line measurements together with multivariate data analysis without time-consuming calibration procedures.     

Biosensor for direct determination of fenitrothion and EPN using recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thiobenzenephosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4-nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citratephosphate buffer, pH 7.5, with 50 μM CoCl₂ at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.     

Reagentless biosensor for phenolic compounds based on tyrosinase entrapped within gelatine film

A simple and new reagentless phenolic compound biosensor was constructed with tyrosinase immobilized in the gelatine matrix cross-linked with formaldehyde. The morphologies of gelatine and gelatine/tryosinase were characterized by SEM. The tyrosinase retains its bioactivity when being immobilized by the gelatine film. Phenolic compounds were determined by the direct reduction of biocatalytically liberated quinone at -0.1 V vs SCE. The process parameters for the fabrication of the enzyme electrode were studied. Optimization of the experimental parameters has been performed with regard to pH, operating potential, temperature and storage stability. This biosensor exhibits a fast amperometric response to phenolic compounds. The linear range for catechol, phenol, and p-Cresol determination was from 5×10⁻⁸ to 1.4×10⁻⁴ M, 5×10⁻⁸ to 7.1×10⁻⁵ M, and 1×10⁻⁷ to 3.6×10⁻⁵ M, with a detection limit of 2.1×10⁻⁸ M, 1.5×10⁻⁸ M, and 7.1×10⁻⁸ M, respectively. The enzyme electrode retained ca.77% of its activity after 7 days of storage at 4°C in a dry state. The proposed sensor presented good repeatability, evaluated in terms of relative standard deviation (R.S.D.=8.6%) for eight different biosensors and was applied for determination in water sample. The recovery for the sample was from 99.0% to 99.8%.     

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs⁻¹ in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75–400 μM and 0.5 μM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C.     

Development of an amperometric biosensor based on covalent immobilization of tyrosinase on a boron-doped diamond electrode

An amperometric enzyme electrode based on direct covalent immobilization of tyrosinase on a boron-doped diamond (BDD) electrode has been developed for the detection of phenolic compounds. Combined chemical and electrochemical modifications of the BDD film with 4-nitrobenzenediazonium tetrafluoroborate, an aminophenyl-modified BDD (AP–BDD) surface was produced, and then the tyrosinase was covalently immobilized on the BDD surface via carbodiimide coupling. The response dependences of the enzyme electrode (Tyr–AP–BDD electrode) on pH of solution, applied potential, oxygen level and phenolic compounds diffusion were studied. The Tyr–AP–BDD electrode shows a linear response range of 1–200, 1–200 and 1–250 μM and sensitivity of 232.5, 636.7 and 385.8 mA M⁻¹ cm⁻² for phenol, p-cresol and 4-chlorophenol, respectively. 90 percent of the enzyme activity of the Tyr–AP–BDD electrode is retained for 5 weeks storing in 0.1 M PBS (pH 6.5) at 4 °C.