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.     

Pure Organic Phase Phenol Biosensor Based on Tyrosinase Entrapped in a Vapor Deposited Titania Sol‐Gel Membrane

A novel amperometric biosensor was constructed for the determination of phenols in pure organic phase. This biosensor was fabricated by immobilizing tyrosinase in a titania sol‐gel membrane which was obtained with a vapor deposition method. This method was facile and avoided the calcination step needed in conventional titania sol‐gel process. The titania sol‐gel membrane could effectively retain the essential water layer around the enzyme molecule needed for maintaining its activity in organic phase. The experimental parameters such as solvent and operating potential were optimized. At ?100 mV this biosensor showed a good amperometric response to phenols in pure chloroform without any mediator and rehydration of the enzyme. For catechol determination the sensor exhibited a fast response of less than 5 seconds. The sensitivity of different phenols was as follows: catechol > phenol > p‐cresol. Additionally, the apparent Michaelis‐Menten constants of the encapsulated tyrosinase to catechol, phenol and p‐cresol were found to be 0.15±0.003, 0.17±0.008 and 0.21±0.004 mM, respectively. The biosensor had also good reproducibility and stability. This work provided a promising platform for the construction of pure organic phase biosensors and the determination of substrates with poor water solubility.     

An Analysis of Mixtures Using Amperometric Biosensors and Artificial Neural Networks

This paper presents a sensor system based on a combination of an amperometric biosensor acting in batch as well as flow injection analysis with the chemometric data analysis of biosensor outputs. The multivariate calibration of the biosensor signal was performed using artificial neural networks. Large amounts of biosensor calibration as well as test data were synthesized using computer simulation. Mathematical and corresponding numerical models of amperometric biosensors have been built to simulate the biosensor response to mixtures of compounds. The mathematical model is based on diffusion equations containing a non-linear term related to Michaelis–Menten kinetics of the enzymatic reaction. The principal component analysis was applied for an optimization of calibration data. Artificial neural networks were used to discriminate compounds of mixtures and to estimate the concentration of each compound. The proposed approach showed prediction of each component with recoveries greater that 99% in flow injection as well as in batch analysis when the biosensor response is under diffusion control.     

Calibration transfer strategy to compensate for instrumental drift in portable quadrupole mass spectrometers

We report the use of a calibration transfer strategy to correct for drift in the quantitative sensitivity of a portable quadrupole mass spectrometer (QMS) aimed at process monitoring applications. Gas mixtures of CH₄/Ar/C₂H₆/CO₂ were studied with calibration phase measurements made of the pure gases for a univariate analysis and of 40 multi-component mixtures for a multivariate approach. To evaluate calibrations, test set spectra of a CH₄/Ar/C₂H₆/CO₂ gas mixture were recorded bi-weekly over a period of 12 months. As part of the strategy a standard of pure argon was measured during both calibration and test phases so that correction factors could be calculated for each measurement day. It was shown that in the absence of a calibration transfer strategy quantifications of test set spectra could be inaccurate by more than an order of magnitude over 12 months. Furthermore, due to the effects of drift in the sensitivity over the 6 days required to record the training set in the calibration phase it was found that the multivariate analysis quantified test spectra less accurately than the univariate analysis. However, by applying the calibration transfer strategy across all measurements (both calibration and test phases) it was shown that the errors in prediction using the multivariate analysis previously seen after 2 weeks were not observed until approximately 12 months later.     

Conductometric tyrosinase biosensor for the detection of diuron, atrazine and its main metabolites

The determination of diuron, atrazine, desisopropylatrazine (DIA) and desethylatrazine (DEA) were investigated using conductometric tyrosinase biosensor. Tyrosinase was immobilised on the biosensor sensitive part by allowing it to mix with bovine serum albumin (BSA) and then cross-linking in saturated glutaraldehyde (GA) vapour for 30 min. The determination of pollutants in a solution was performed by comparison of the output signal (i.e percentage of the enzymatic activity) of the biosensor before and after contact with pollutants. The measurement of the enzymatic activity was performed using 4-chlorophenol, phenol and catechol substrates and response times ranging from 1 to 5 min were observed. A 4-chlorophenol substrate was used to detect pesticides. A 30 min contact time of the biosensor in the pollutant solution was used. Under the experimental conditions employed, detection limits for diuron and atrazine were about 1 ppb and dynamic range of 2.3-2330 and 2.15-2150 ppb were obtained for diuron and atrazine, respectively. A relative standard deviation (n=3) of the output signal was estimated to be 5% and a slight drift of 1.5 μS h⁻¹ was observed. The 90% of the enzyme activity was still maintained after 23 days of storage in a buffer solution at 4 °C.     

Amperometric Phenol Biosensor Based on Horseradish Peroxidase Entrapped PVF and PPy Composite Film Coated GC Electrode

Polyvinylferrocene (PVF) was used as a mediator for the fabrication of a horseradish peroxidase (HRP)-modified electrode to detect phenol derivatives via a composite polymeric matrix of conducting polypyrrole (PPy). Through an electropolymerization process, enzyme HRP was entrapped with PPy in a three-electrode system onto a glassy carbon electrode previously covered with PVF, resulting in a composite polymeric matrix. Steady-state amperometric measurements were performed at ?200 mV vs. Ag/AgCl in aqueous phosphate buffer containing NaCl 0.1 M (pH 6.8) in the presence of hydrogen peroxide. The response of the HRP-modified PVF electrode was investigated for various phenol derivatives, which were 4-chlorophenol, phenol, catechol, hydroquinone, 2-aminophenol, pyrogallol, m-cresol, and 4-methoxyphenol. Analytical parameters for the fabricated PVF electrode were obtained from the calibration curves. The highest sensitivity was obtained from the calibration of 4-chlorophenol as 29.91 nA/μM. The lowest detection limit was found to be 0.22 μM (S/N?=?3) for catechol, and the highest detection limit was found to be 0.79 μM (S/N?=?3) for 4-methoxyphenol among the tested derivatives. The biosensor can reach 95% of steady-state current in about 5 min. The electrode is stable for 2 months at 4 °C.     

Diazirine‐functionalized Nanostructured Platform for Enzymes Photografting and Electrochemical Biosensing

A simple technique for the construction of a versatile diazirine‐functionalized nanostructured platform for enzymes photografting and electrochemical biosensing was proposed in this work. The feasibility of the approach was proved by photo crosslinking of an enzyme, tyrosinase, to diazirine‐activated aminated carbon nanotubes coated glassy carbon electrode. The analytical performances of the realized biosensor were evaluated employing catechol as analyte. Then the sensor based on the diazirine‐functionalized nanostructured platform with photografted tyrosinase was applied together with the high resolution technique Differential Alternative Pulses Voltammetry for dopamine determination in the linear concentration range of 5–25 μmol L^?1 in the presence of interfering agents as uric acid up to its 100‐fold excess.     

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.     

Reassessment of the calibration constant for the IAsys biosensor.

A magnitude of 50 are s ng-1 mm2 has been determined for the calibration constant relating biosensor response to the amount of protein bound to the sensor surface of an IAsys cuvette. These studies entailed enzymatic assessment of the extent of lactate dehydrogenase depletion in the liquid phase arising from enzyme binding to a carboxymethyldextran-coated sensor surface, and also estimation of a maximum biosensor response for the electrostatic interaction of ovalbumin with an aminosilane-coated sensor surface. The latter results required correction for contributions to biosensor response resulting from changes in the refractive index of the liquid phase effected by high protein concentrations.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Development and optimization of a solid composite tyrosinase biosensor for phenol detection in flow injection systems

Bulk-modified epoxy-graphite tyrosinase biosensors were fabricated by four different procedures. The influence of these fabrication procedures on the analytical performance of the enzyme electrode in an amperometric wall-jet flow cell has been studied. The bioprobe performance is assessed by cyclic voltammetry. Higher current densities and narrower peaks were obtained when the enzyme was introduced in the dry state into the epoxy-graphite material, instead of introducing it previously dissolved in the buffer. In the F1 system responses of 11.79 μA cm⁻² and 1.43 μA cm⁻² are then obtained for catechol and phenol respectively for 50 μL injections of 20 μM solutions. Moreover, if gold/palladium is introduced into the epoxy-graphite, a further increase in current is achieved resulting in 27.70μA cm⁻² and 4.90μA cm⁻²for catechol and phenol, respectively. This biosensor can operate in aqueous as well as in mixed aqueous-organic environments.     

Amperometric sensors based on tyrosinase-modified screen-printed arrays

This paper describes the design, development and characteristics of a tyrosinase (polyphenol oxidase) modified amperometric screen-printed biosensor array, with the enzyme cross-linked in a redox-hydrogel namely the PVI₁₃-dmeOs polymer. Two types of Au-screen-printed four-channel electrode arrays, differing in design and insulating layer, were compared and investigated. Au-, graphite-coated-Au- and Carbopack C-coated-Au-surfaces, serving as the basis for tyrosinase immobilisation, were investigated and the performances of the different arrays were evaluated and compared in terms of their electrocatalytic characteristics, as well as operational- and storage stability using catechol as model substrate. It was found that the Carbopack C-coated array was the best choice for tyrosinase immobilisation procedure mainly due to a higher mechanical stability of the deposited enzyme layer, combined with good sensitivity and stability for up to 6 months of use. In the batch mode the biosensors responded linearly to catechol up to 30 μM with limits of detection from 0.14 μM. Parameters from cyclic voltammograms indicated that the reversibility of the direct electrochemical reaction for catechol on the three types of electrode surfaces (no tyrosinase modification) was not the limiting factor for the construction and performance of tyrosinase biosensors.     

Multivariate Calibration Transfer between two Potentiometric Multisensor Systems

A multivariate calibration of multisensor systems is a very important stage in their application. Construction of reliable calibration model requires significant investment of time and money. Once established, calibration model can be applied normally only together with that particular multisensor system, which was employed for its’ construction – this can be a serious limitation for a wide adoption of multisensor systems in common laboratory practice. In order to address this issue we have studied the applicability of several calibration transfer techniques, such as direct standardization, single wavelength standardization and standardization with regularization coefficient for the data obtained from two potentiometric multisensor systems in analysis of complex lanthanide mixtures. It was found that mathematical correction of sensor array response using standardization with regularization coefficient allows for using the regression model derived for one sensor array together with the data obtained from another sensor array. The value of root mean squared error of prediction for total lanthanide concentration increased insignificantly (0.10 instead of 0.07 in log scale) compared with that provided by the first multisensor system.     

Application of a Partial Least Squares Regression for the Determination of Nanomolar Concentrations of Scandium in the Presence of Nickel by Adsorptive Stripping Voltammetry

This paper presents the methodology of a very sensitive determination of scandium in excess of nickel by adsorptive stripping voltammetry on a mercury film electrode and PLS regression. A calibration set consisting of binary mixtures containing 5, 15, 25, 35 or 45×10^?9 M Sc(III) and simultaneously 0.5–50×10^?7 M of Ni(II) was used to develop the chemometric PLS calibrations. An external set containing synthetic mixtures of 10, 20, 30, 40×10^?9 M Sc(III) and the same Ni(II) concentration as mentioned above was used to validate the model and evaluate predictive ability. The application of data pretreatment techniques involving baseline correction, smoothing, range‐scaling, mean‐centering and their influence on the PLS model complexity, were also investigated. In the effect, the model for Sc(III), including 6 latent variables, was constructed. The model fulfills validation criteria and is characterized by a good prediction ability (majority of the prediction errors are lower than 10%). This work shows significant progress in the development of a very sensitive analytical technique for the determination of scandium in the presence of different concentrations of nickel by application of multivariate calibration tools.     

Simultaneous Spectrophotometric Determination of Benzyl Alcohol and Diclofenac in Pharmaceutical Formulations by Chemometrics Method

Diclofenac sodium (DS) is a drug with analgesic, antipyretic, and anti‐inflammatory properties. It is present in numerous pharmaceutical preparations. In injectable forms, it is usually accompanied by benzyl alcohol (BA) as an excipient, which is used as a blocking anesthetic (4%) and an antiseptic (4–10%). In this work a spectrophotometric methodology was applied in order to determine benzyl alcohol and diclofenac in injectable formulations by applying a multivariate calibration method. By a multivariate calibration method such as partial least squares (PLS), it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. In this study, the concentration model is based on absorption spectra in the 230–320 nm range for 25 different mixtures of benzyl alcohol and diclofenac. Calibration matrix contains 10–95 and 1–50 μg mL^?1 for benzyl alcohol and diclofenac, respectively. The root mean square errors of prediction (RMSEP) for benzyl alcohol and diclofenac were 3.0776 and 1.7557, respectively. The proposed method was validated by using a set of synthetic sample mixtures and subsequently applied to simultaneous determination of benzyl alcohol and diclofenac in two different pharmaceutical formulations.     

Spectrophotometric simultaneous determination of ceratine, creatinine, and uric acid in real samples by orthogonal signal correction–partial least squares regression

Abstract  This work describes a quantitative spectroscopic method for the analysis of ternary mixtures of ceratine (CER), creatinine (CRE), and uric acid (UA) using multivariate data models based upon ultraviolet spectroscopy. By multivariate calibration methods, such as partial least squares regression, it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. In this study, the calibration model is based on absorption spectra in the 200–260 nm range for 36 different mixtures of CER, CRE, and UA. The unrelated information was removed by the orthogonal signal correction (OSC) method and the results were proved. Evaluation of the prediction errors for the prediction set reveals the OSC-treated data give substantially lower root mean square error of prediction (RMSEP) values than original data. The RMSEP for CER, CRE, and UA with OSC were 1.1686, 0.2195, and 0.3726, and without OSC were 1.9057, 0.3482, and 0.6164, respectively. This procedure allows the simultaneous determination of CER, CRE, and UA in synthetic and real samples. Graphical abstract        

Application of polypyrrole nanowires for the development of a tyrosinase biosensor

Polypyrrole nanowires (PPyNWs) were fabricated and examined as a structural component of amperometric biosensor matrix. An enzyme, tyrosinase (TYR), was immobilized onto PPyNWs using glutaraldehyde (GA). Matrix composite morphology was investigated using scanning electron microscopy. Electrochemical behavior of the prepared PPyNWs/GA/TYR biosensor towards catechol was studied and the assessment of its analytical characteristics was carried out taking into account linear range, sensitivity, repeatability, reproducibility and operational stability.     

Amperometric Flow‐Injection Determination of Phenolic Compounds Using a Biosensor with Immobilized Laccase,Peroxidase and Tyrosinase

A screen‐printed four‐electrode sensor based on immobilization of laccase (Coriolus hirsutus), peroxidase (horseradish) and tyrosinase (mushroom) in the same array was developed for monitoring of phenols. The enzymes were immobilized onto a self‐assembled monolayer (4‐mercapto‐1‐butanol) modified gold surface via covalent attachment by epichlorohydrin coupling. The experimental conditions for simultaneous operation of the three enzymes were optimized based on catechol determination. The sensors were further applied for the amperometric detection of several substituted phenolic compounds, carried out using a single line flow‐injection system. Hydrogen peroxide served as co‐substrate for peroxidase. The limits of detection for phenols in aqueous solutions were in the micromolar range, one assay was completed in less than 5 min. The preliminary studies showed that the compatibility of the above mentioned enzyme array enabled the multielectrode biosensor to be applied to real samples including industrial wastewaters and surface waters.     

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20–40 ng mL⁻¹ and root mean square errors for the validation samples in the range 30–60 ng mL⁻¹ (corresponding to relative prediction errors of 3–8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2–8 ng mL⁻¹, root mean square errors, 3–50 ng mL⁻¹ and relative prediction errors, 1–5%.     

Preparation and characterization of a new enzyme electrode based on solid paraffin and activated graphite particles

A new electrochemical biosensor was developed by incorporating an enzyme into a solid-paraffin-graphite-particle matrix. Tyrosinase served as model enzyme and the biosensor response was characterized with respect to its response to dopamine. The influence of different experimental parameters (tyrosinase loading, flow rate, oxygen dependence, pH, etc.) was investigated in order to optimize the biosensor performance. The electrode response was fast, reversible and linear in a large concentration domain (0.1 muM-1 mM). The enzyme-solid paraffin carbon paste electrode (CPE) showed markedly improved stability in flow injection analysis compared to the classical liquid paraffin-graphite-based biosensors. The biosensor allowed a sampling rate of 79 samples per hour, the repeatability of the injections was improved with respect to the classical CPE with a relative standard deviation of 2.2% (N = 63), and the detection limit for dopamine was 50 nM. The biosensor response to some phenol and catechol derivatives was also investigated.