Laccase-based Biosensor Research Articles

Amperometric biosensor based on Laccase immobilized onto a screen-printed electrode by Matrix Assisted Pulsed Laser Evaporation

A Laccase-based biosensor for the determination of phenolic compounds was developed by using Matrix Assisted Pulsed Laser Evaporation as an innovative enzyme immobilization technique. and the deriving biosensor was characterized and applied for the first time. Laccase was immobilized onto different substrates including screen printed carbon electrodes and spectroscopic, morphologic and electrochemical characterizations were carried out. A linear range from 1 to 60μM was achieved working at 5.5pH and −0.2V detection potential vs Ag pseudoreference. The limits of detection and quantification were found to be 1 and 5μM, respectively. A good fabrication reproducibility, stability of response and selectivity toward interferents were also found The potential of the developed biosensor was tested in the determination of total polyphenol content in real matrices (tea infusion, ethanolic extract from Muscari comosum bulbs and aqueous solution of a food supplement from black radish root and artichoke leaves) and the results were compared with those obtained by using the Folin–Ciocalteu method.

Immobilized laccase-based biosensor for the detection of disubstituted methyl and methoxy phenols – application of Box–Behnken design with response surface methodology for modeling and optimization of performance parameters

An amperometric principle-based biosensor, employing immobilized laccase enzyme from Trametes versicolor, was developed for the detection of disubstituted methyl and methoxy phenols. Three immobilization methods such as entrapment, cross-linking, and co-cross-linking, with bovine serum albumin (BSA) on nylon membrane have been compared. Among tested methods of immobilization, co-cross-linking method with BSA was superior to the other methods in terms of; sensitivity, limit of detection, response time, and operating stability. The increased sensitivity of the probe optimization of concentrations of laccase, BSA and glutaraldehyde can be achieved by, employing the Box–Behnken design of experiment.

Studies on enhancing operational stability of a reusable laccase-based biosensor probe for detection of ortho-substituted phenolic derivatives.

An amperometric principle-based biosensor containing immobilized enzyme laccase from Trametes versicolor was developed for detection of ortho-substituted phenolic derivatives. Different immobilization methods for Trametes versicolor laccase enzyme on cellophane membrane and the enhancement of operational stability of the immobilized enzyme electrode using various protein-based stabilizing agents were studied. Among tested methods of immobilization, co-cross-linking method with bovine serum albumin was superior to the other methods in terms of sensitivity, limit of detection, response time, and operating and thermal stability. Biosensor response reached steady state within 3 min and exhibited maximum activity at 45 °C and pH 6.8. The sensitivity of the ortho-substituted phenols for the test biosensor developed with co-cross-linking method of immobilization using bovine serum albumin as the protein-based stabilizing agent was in the order: 2-aminophenol > guaiacol(2-methoxyphenol) > catechol(2-hydroxyphenol) > cresol(2-methyl phenol) > 2-chlorophenol. Validation of the newly developed biosensor by comparison with HPLC showed good agreement in the results. A newly developed biosensor was applied for quantification of ortho-substituted phenols in simulated effluent samples.

Fabrication of an Amperometric Flow-Injection Microfluidic Biosensor Based on Laccase for In Situ Determination of Phenolic Compounds.

We aim to develop an in situ microfluidic biosensor based on laccase from Trametes pubescens with flow-injection and amperometry as the transducer method. The enzyme was directly immobilized by potential step chronoamperometry, and the immobilization was studied using cyclic voltammetry and electrochemical impedance spectroscopy. The electrode response by amperometry was probed using ABTS and syringaldazine. A shift of interfacial electron transfer resistance and the electron transfer rate constant from 18.1 kΩ to 3.9 MΩ and 4.6 × 10−2 cm s−1 to 2.1 × 10−4 cm s−1, respectively, evidenced that laccase was immobilized on the electrode by the proposed method. We established the optimum operating conditions of temperature (55°C), pH (4.5), injection flow rate (200 µL min−1), and applied potential (0.4 V). Finally, the microfluidic biosensor showed better lower limit of detection (0.149 µM) and sensitivity (0.2341 nA µM−1) for ABTS than previous laccase-based biosensors and the in situ operation capacity.

A catechol biosensor based on electrospun carbon nanofibers.

Carbon nanofibers (CNFs) were prepared by combining electrospinning with a high-temperature carbonization technique. And a polyphenol biosensor was fabricated by blending the obtained CNFs with laccase and Nafion. Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscope (FE-SEM) were, respectively, employed to investigate the structures and morphologies of the CNFs and of the mixtures. Cyclic voltammetry and chronoamperometry were employed to study the electrocatalysis of the catechol biosensor. The results indicated that the sensitivity of the biosensor was 41 µA·mM−1, the detection limit was 0.63 µM, the linear range was 1–1310 µM and the response time was within 2 seconds, which excelled most other laccase-based biosensor reported. Furthermore, the biosensor showed good repeatability, reproducibility, stability and tolerance to interferences. This novel biosensor also demonstrated its promising application in detecting catechol in real water samples.

Simple laccase-based biosensor for formetanate hydrochloride quantification in fruits

This work describes the development of an electrochemical enzymatic biosensor for quantification of the pesticide formetanate hydrochloride (FMT). It is based on a gold electrode modified with electrodeposited gold nanoparticles and laccase. The principle behind its development relies on FMT's capacity to inhibit the laccase catalytic reaction that occurs in the presence of phenolic substrates. The optimum values for the relevant experimental variables such as gold nanoparticles electrochemical deposition (at −0.2V for 100s), laccase immobilization (via glutaraldehyde cross-linking), laccase concentration (12.4mg/mL), substrate selection and concentration (5.83×10−5M of aminophenol), pH (5.0), buffer (Britton–Robinson), and square-wave voltammetric parameters were determined. The developed biosensor was successfully applied to FMT determination in mango and grapes. The attained limit of detection was 9.5×10−8±9.5×10−10M (0.02±2.6×10−4mg/kg on a fresh fruit weight basis). Recoveries for the five tested spiking levels ranged from 95.5±2.9 (grapes) to 108.6±2.5% (mango). The results indicated that the proposed device presents suitable characteristics in terms of sensitivity (20.58±0.49A/μM), linearity (9.43×10−7 to 1.13×10−5M), accuracy, repeatability (RSD of 1.4%), reproducibility (RSD of 1.8%) and stability (19days) for testing of compliance with established maximum residue limits of FMT in fruits and vegetables.

Rapid Screening Method for Assessing Total Phenolic Content Using Simple Flow Injection System with Laccase based-biosensor

Rapid Screening Method for Assessing Total Phenolic Content Using Simple Flow Injection System with Laccase based-biosensor

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi-walled carbon nanotubes (MWCNTs) paste electrode modified by dispersion of laccase (3%, w/w) within the optimum composite matrix (60:40%, w/w, MWCNTs and paraffin binder) showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate 4-aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90×10−7 to 1.15×10−5molL−1 using 4-aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%). The limit of detection obtained was 1.8×10−7molL−1 (0.04mgkg−1 on a fresh weight vegetable basis). The high activity and catalytic properties of the laccase-based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0±0.3% were attained. No significant effects in the pirimicarb electroanalysis were observed by the presence of pro-vitamin A, vitamins B1 and C, and glucose in the vegetable extracts. The proposed biosensor-based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Development of optical laccase-based biosensors for phenolic compound detection

Development of optical laccase-based biosensors for phenolic compound detection

Herbal infusions bioelectrochemical polyphenolic index: Green tea – The gallic acid interference

An amperometric biosensor containing immobilised laccase from Trametes versicolor was used for the quantification of phenolic compounds in herbal infusions and green tea samples, from nine botanical families. The main purpose of this research was to correlate the bioelectrochemical polyphenolic index (BPI) of the samples with the total phenolic content according to the Folin–Ciocalteu spectrophotometric method (TPC), and the total antioxidant activity (TAA) measured by TEAC (Trolox Equivalent Antioxidant Capacity). A strong correlation between BPI, TPC, and TAA was obtained for 10 herbal infusion samples. However, when a green tea sample was taken into account, a decrease in the linear correlation coefficient (r2) from 0.9949 to 0.2599 and 0.5609 to 0.1086 for the relationship between BPI/TPC and BPI/TAA, respectively, was observed. Thus, we could conclude that the green tea’s matrix affects the laccase-based biosensor response. HPLC–DAD analysis showed the presence of gallic acid only in the green tea sample. The results have indicated that gallic acid inhibited the laccase activity regarding the ABTS oxidation in a concentration-dependent manner. The very strong correlation between BPI/TPC obtained for herbal infusions allow us to conclude that the laccase-based biosensor, used in this research, provides a valuable tool to obtain a valid estimation of the classical Folin–Ciocalteu index, in an uncomplicated and fast way.

Laccase-Nafion Based Biosensor for the Determination of Polyphenolic Secondary Metabolites

A laccase-based biosensor was developed by specific enzyme adsorption on screen-printed working electrodes of DROPSENS cells, and stabilized with Nafion 0.1% membrane. The electrode was characterized with respect to response time, sensitivity, linear range, detection limit, pH dependence, interferences, and long-term stability. The tested substrates were catechol, rosmarinic acid, caffeic acid, chlorogenic acid, and gallic acid. The optimized biosensor proved the following characteristic performances: the apparent Michaelis Menten calculated considering rosmarinic acid substrate 8.3 × 10−6 mol L−1 (r = 0.995, n = 6); the dynamic range of biosensor response for rosmarinic acid 7 × 10−7 − 1.5 × 10−6 mol L−1; the detection limit for rosmarinic acid 1.19 × 10−7 mol L−1 (RSD = 1.08%, n = 3). It was noticed that the biosensor reaches systematically 90% to 94.3% from the response obtained by LC-DAD-ESI-MS for real samples.

Evaluating the antioxidant capacity of wines: a laccase-based biosensor approach

Polyphenolic compounds present in wine have a high antioxidant capacity providing a protective effect on human health. There is a worldwide agreement on this assertion. However, there is no agreement on the method of assessing the antioxidant capacity of wines. The purpose of this study was to emphasize the ability of the laccase-based biosensor developed by our group to evaluate the antioxidant capacity of several types of wine in relation to their polyphenolic content. The values of total antioxidant activity of the investigated wine samples were well correlated with the corresponding bioelectrochemical polyphenol-estimated values, the correlation coefficient obtained being r = 0.9795 (p < 0.05). The linear relationship between total antioxidant activity and bioelectrochemical polyphenol index determined by laccase-based biosensor enabled the calculation of the TEAC (Trolox Equivalent Antioxidant Capacity) of a wine from its bioelectrochemical polyphenol index.

Laccase-based biosensor for the determination of polyphenol index in wine

In this work we have developed and characterized the use of Laccases from Trametes versicolor (TvL) and Trametes hirsuta (ThL) as biocatalytic components of electrochemical biosensors for the determination of polyphenol index in wines. Polyazetidine prepolimer (PAP) was used as immobilizing agent, multi-walled and single-walled carbon nanotubes screen-printed electrodes as sensors (MWCNTs-SPE and SWCNTs-SPE) and gallic acid as standard substrate. The amperometric measurements were carried out by using a flow system at a fixed potential of -100 mV vs. silver/silver chloride electrode in Britton-Robinson buffer 0.1 mol L(-1), pH 5. The results were compared with those obtained with the Folin-Ciocalteau reference method. The results obtained in the analysis of twelve Italian wines put in evidence the better suitability of ThL-MWCNTs-based biosensor in the determination of the polyphenol index in wines. This biosensor shows fast and reliable amperometric responses to gallic acid with a linear range 0.1-18.0 mg L(-1) (r(2)=0.999). The influence of the interferences on both spectrophotometric and electrochemical measurements have been carefully evaluated.

Laccase-based biosensors for monitoring lignin

Four different ways to detect Kraft and soluble pine lignin using either soluble or immobilised Trametes hirsuta laccase have been investigated. In the first, the concentration of lignin was measured using laccase in homogeneous medium with a Clark electrode. In the second, laccase immobilised onto aminated porous glass beads was used in a reactor submerged into the lignin containing solution and the reaction was followed with a Clark electrode. The third and fourth techniques involved lignin detection using laccase modified spectrographic graphite electrodes, either used in flow injection mode or in batch mode, where lignin solutions were injected into an electrochemical cell. It was shown that the use of laccase modified graphite electrodes in conjunction with flow injection for amperometric lignin detection is an attractive perspective and our data form the basis for designing laccase-based biosensors to be applied for measuring lignin and its model compounds in waste water, e.g. from the pulp and paper industry.

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.

Electrochemical biosensor-based devices for continuous phenols monitoring in environmental matrices

A flow system method for continuous determination of phenolic compounds in environmental matrices was employed using a dialysis membrane sampler and laccase- and tyrosinase-based biosensors as detector. The biosensors response to different phenolic compounds was investigated. The laccase-based biosensor showed high sensitivity to guaiacol and chloroguaiacol, while the tyrosinase-based biosensor was more sensitive to phenol and chlorophenol. Both of the biosensors presented highly selective measurements of micromolar concentration of these compounds. Detection limits around 1.1 x 10-7 mol L-1 for guaiacol and 1.9 x 10-7 mol L-1 for chloroguaiacol could be estimated for the laccase-based biosensor. Using the tyrosinase-based biosensor detection limits of 1.5 x 10-7 mol L-1 for phenol and 9.0 x 10-8 mol L-1 for chlorophenol were observed. The proposed flow method presented a linear response range between 1.0 mmol L-1 and 100.0 mmol L-1, in the optimized operational conditions (laccase-based biosensor: pH 5.0 and 0 mV vs Ag/AgCl as working potential; tyrosinase-based biosensor: pH 5.5 and 50 mV vs Ag/AgCl as working potential). Real paper mill effluent sample was analyzed by this system and by the reference colorimetric method and the results were discussed.

Development of a laccase-based flow injection electrochemical biosensor for the determination of phenolic compounds and its application for monitoring remediation of Kraft E1 paper mill effluent

This paper describes the development of a new system for amperometric determination of phenolic compounds, and its application for monitoring these compounds in paper mill effluent. The method was based on a flow system, a dialysis sampler, and a laccase-based biosensor. The performance of this system was investigated with respect to pH, ionic strength, working potential, and flow-rate dependence. The biosensor showed an excellent long-term stability allowing measurements for over than 3 months. The sensitivity of laccase-based biosensor was tested for phenol, p-chlorophenol, guaiacol and chloroguaiacol; the detector presented selective measurements of micromolar concentration of these compounds. The integration of a dialysis membrane sampling in the system protected the biosensor surface from fouling and gave independence of sample conditions that commonly influence the biosensor performance. These favorable characteristics allowed its application for direct measurements in complex media with no sample pretreatment. This ability was confirmed employing this system in a continuous analysis of phenolic compounds during the remediation of paper mill effluent by ozonization process.

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practial validity of the method was demonstrated by tannin analysis in tea of different brands.