Laccase-based Biosensor Research Articles

Electrospray deposition of starch-containing laccase: A green technique for low-cost and eco-friendly biosensors

Recently a laccase-based biosensors with unprecedented reuse and storage capabilities in the detection of catechol compound has been manufactured using ambient Electrospray Deposition (ESD) technique. These biosensors showed to be reused up to 63 measurements on the same electrode just prepared at room temperature and pressure. In this new work the reasons behind such a high-performance functioning have been investigated by analysing the commercial sample of laccase with different chemical physics methods: Electrophoresis, Fourier Transform Infrared Spectroscopy, X-ray Fluorescence and Nuclear Magnetic Resonance Spectroscopy. The analyses reveal the presence of the starch in the sample and its essential role as stabilizing agent. Indeed, comparing the performance of starch/laccase-based biosensors with starch-free/laccase-based biosensors, both produced via ESD, showed that the starch-free biosensors lost about 40% of their performance after just the first wash. This suggests that the presence of starch in the laccase sample is a key factor in providing the high wash and storage resistance, which are essential for the fabrication of such devices.

Determination of the total phenolic content in Hardaliye by amperometric laccase-based biosensor and comparison of the LC-MS/MS

In this study, a laccase-based amperometric biosensor was fabricated for the quantitative analysis of total phenolic content in Hardaliye. The biosensor was constructed using a carbon paste electrode with simple modifications. The laccase obtained from Trametes versicolor was immobilized into a carbon paste electrode modified with a gelatin matrix. The immobilization parameters for biosensor fabrication, as well as its working conditions were optimized with gallic acid as the phenolic standard. Additionally, molecular docking simulation was used to examine the interaction between Trametes versicolor laccase and gallic acid. The biosensor's analytical parameters were determined under optimal conditions including linearity, repeatability, stability and selectivity. The linear range of gallic acid was calculated as 0.5–100 μM, with the detection limit of 0.1323 ± 0.1 μM. With good selectivity and stability, the biosensor successfully determined the total phenolic contents in Hardaliye, and red-wine samples. Comparison studies showed that the biosensor results agreed with those obtained from the Liquid Chromatography-Tandem Mass Spectrometry method. This work indicates that the proposed biosensor is a promising analytic tool for total phenolic analyses of beverages.

Biosensing L-DOPA with laccase-based enzyme electrodes: a comparative study

The focus of the present work is to develop and optimize an electrochemical laccase-based biosensor for the determination of 3,4-dihydroxyphenyl-alanine, also known as L-DOPA. The biosensor was assembled on a conventional glassy carbon electrode, the surface of which was covered with laccase enzyme retained under a thin Nafion™ membrane. The enzymes used for this purpose were isolated and purified from the white-rot basidiomycetes (Trametes sp.) Trametes pubescens and Trametes versicolor. Although biochemically similar, the two enzymes demonstrated some differences in their affinity not only using 3,4-dihydroxyphenyl-L-alanine as enzyme substrate, but also when catecholamines such as dopamine and L-epinephrine were used. A range of electrochemical techniques were used for the study, such as cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. Experiments were performed using buffers with different pH and applying various substrate concentrations. Activity and sensitivity of the two alternative laccase – based biosensors were compared by means of chronoamperometry. The biosensor produced on the basis of Trametes pubescens laccase, operating in citrate buffer with pH 4 proved to be more suitable than the one based on laccase purified from Trametes versicolor for biosensing L-DOPA.

Influence of different covalent immobilization protocols on electroanalytical performance of laccase-based biosensors

This paper presents a simple and effective flow-through electrochemical biosensor, consisting of Trametes versicolor laccase (Lac)-based mini-reactor and a tubular detector of silver solid amalgam (TD-AgSA), capable of rapid and selective detection of phenolic compounds. Amperometric detection relies on the reduction of the quinone molecule (formed during the enzymatic reaction in a mini-reactor) on TD-AgSA at –50 mV vs SCE. Since different enzyme immobilization techniques may contribute to differing biosensor performances, four covalent strategies for Lac attachment were compared: (i) through glutaraldehyde to supports −NH2, (ii) via disuccinimidyl suberate to supports −NH2, (iii) using EDC/NHS for Lac coupling by its −COOH groups to supports −NH2, and (iv) using EDC/NHS to supports −COOH. Additionally, five supports (mesoporous silica (SBA-15, MCM-41), cellulose, carbon-based (glassy carbon, graphite) powders) were investigated. It was found that different methods of immobilization, as well as different types of supports, significantly affect the amount of immobilized Lac and, in turn, the analytical characteristics of the obtained biosensors. Thus, TD-AgSA with enzymatic mini-reactor based on Lac covalently attached via glutaraldehyde to aminated MCM-41 proved to be the most promising biosensor, providing the best detection limit (18.3 μmol L–1) and the long-term stability (47.9 % of the initial response/4 months/100 measurements).

Biosensor based on laccase-halloysite nanotube and imidazolium zwitterionic surfactant for dopamine determination

Zwitterionic surfactants incorporated nanomaterials and biomolecules structures offers enhanced catalytic activities and stabilities in biosensing. Herein, we present the construction of biosensors using graphite, a class of imidazolium zwitterionic surfactants, 3-(1-alkyl-3-imidazolio)-propane-sulfonate (ImS3-n; n = 10, 12, 14, 16) and laccase (Lac) immobilized on clay halloysite nanotube (HNT) in a carbon paste electrode (CPE). The Lac-HNT-ImS3–14/CPE showed to be the most promising biosensor due to the low capacitance and fastest electronic charge transfer using the probe hexacyanoferrate. Therefore, the surfactant ImS3–14 was characterized by spectroscopic techniques such as 1H, 13C NMR and ESI-MS. The developed biosensor was used to create a novel electroanalytical method for the determination of dopamine by square-wave voltammetry (SWV). Under optimized conditions, the calibration plot showed a linear range for dopamine of 0.99–67.8 µmol L−1 using SWV. The limit of detection calculated was 0.252 µmol L−1. The biosensor was successfully applied in the determination of dopamine contained in pharmaceutical samples, and the new proposed sensor was obtained with a recovery range from 97.6% to 108.7 % (n = 3). The Lac-HNT-ImS3–14/CPE biosensor is presented here as a new laccase-based biosensor with outstanding characteristics, which offered an elegant way for interfacing the electrochemical biosensor at the molecular level. It also plays a significant role in electronic signal transduction and biological recognition processes.

Recent Advances in the Development of Laccase-Based Biosensors via Nano-Immobilization Techniques

Monitoring phenolic compounds is critical in the environmental, food, and medical sectors. Among many recent advanced detection platforms, laccase-based biosensing platforms gave very rapid, effective, online, and in situ sensing of phenolic compounds. In laccase-based biosensors, laccase immobilization techniques have a vital role. However, a detailing of the advancements in laccase immobilization techniques employed in laccase-based biosensors is lacking in the literature. Thus, in this review, we assessed how the nano-immobilization techniques shaped the laccase biosensing platforms. We discussed novel developments in laccase immobilization techniques such as entrapment, adsorption, cross-linking, and covalent over new nanocomposites in laccase biosensors. We made a comprehensive assessment based on the current literature for future perspectives of nano-immobilized laccase biosensors. We found the important key areas toward which future laccase biosensor research seems to be heading. These include 1. A focus on the development of multi-layer laccase over electrode surface, 2. The need to utilize more covalent immobilization routes, as they change the laccase specificity toward phenolic compounds, 3. The advancement in polymeric matrices with electroconductive properties, and 4. novel entrapment techniques like biomineralization using laccase molecules. Thus, in this review, we provided a detailed account of immobilization in laccase biosensors and their feasibility in the future for the development of highly specific laccase biosensors in industrial, medicinal, food, and environmental applications.

Fabrication of a New, Low-Cost, and Environment-Friendly Laccase-Based Biosensor by Electrospray Immobilization with Unprecedented Reuse and Storage Performances.

The fabrication of enzyme-based biosensors has received much attention for their selectivity and sensitivity. In particular, laccase-based biosensors have attracted a lot of interest for their capacity to detect highly toxic molecules in the environment, becoming essential tools in the fields of white biotechnology and green chemistry. The manufacturing of a new, metal-free, laccase-based biosensor with unprecedented reuse and storage capabilities has been achieved in this work through the application of the electrospray deposition (ESD) methodology as the enzyme immobilization technique. Electrospray ionization (ESI) has been used for ambient soft-landing of laccase enzymes on a carbon substrate, employing sustainable chemistry. This study shows how the ESD technique can be successfully exploited for the fabrication of a new promising environment-friendly electrochemical amperometric laccase-based biosensor, with storage capability up to two months without any particular care and reuse performance up to 63 measurements on the same electrode just prepared and 20 measurements on the one-year-old electrode subjected to redeposition. The laccase-based biosensor has been tested for catechol detection in the linear range 2–100 μM, with a limit of detection of 1.7 μM, without interference from chrome, cadmium, arsenic, and zinc and without any memory effects.

Laccase and Tyrosinase Biosensors Used in the Determination of Hydroxycinnamic Acids.

In recent years, researchers have focused on developing simple and efficient methods based on electrochemical biosensors to determine hydroxycinnamic acids from various real samples (wine, beer, propolis, tea, and coffee). Enzymatic biosensors represent a promising, low-cost technology for the direct monitoring of these biologically important compounds, which implies a fast response and simple sample processing procedures. The present review aims at highlighting the structural features of this class of compounds and the importance of hydroxycinnamic acids for the human body, as well as presenting a series of enzymatic biosensors commonly used to quantify these phenolic compounds. Enzyme immobilization techniques on support electrodes are very important for their stability and for obtaining adequate results. The following sections of this review will briefly describe some of the laccase (Lac) and tyrosinase (Tyr) biosensors used for determining the main hydroxycinnamic acids of interest in the food or cosmetics industry. Considering relevant studies in the field, the fact has been noticed that there is a greater number of studies on laccase-based biosensors as compared to those based on tyrosinase for the detection of hydroxycinnamic acids. Significant progress has been made in relation to using the synergy of nanomaterials and nanocomposites for more stable and efficient enzyme immobilization. These nanomaterials are mainly carbon- and/or polymer-based nanostructures and metallic nanoparticles which provide a suitable environment for maintaining the biocatalytic activity of the enzyme and for increasing the rate of electron transport.

Effect of Multi-Walled Carbon Nanotubes on Analytical Parameters of Laccase-Based Biosensors Received by Soft Plasma Polymerization Technique

Development of new biosensors is currently an important issue of science and industry. The latest achievement in biosensing technology is the application of cold, non-equilibrium plasma in the form of Corona Discharges for the deposition of bio-active recognition layers. The innovative SPP (Soft Plasma Polymerization) technique allows to prepare Laccase-based biosensor with comparable dynamic range and sensitivity as devices received by conventional “wet” methods. The main purpose of this work was to investigate the effect of multi-walled carbon nanotubes (MWCNT) on analytical parameters, working conditions and applicability of Laccase-based biosensors constructed by SPP technique. The received results show that Laccase-based biosensor containing MWCNT interlayer exhibited two linear ranges from 0.03 to 0.2 μM and from 0.2 to 3.0 μM with high correlation coefficients. Moreover, the biosensor can be successfully used in pharmaceutical industry because both common foreign substances as well as matrixes of three pharmaceutical samples did not influence on analytical signal generated by the biosensor.

Laccase-Based Biosensor Encapsulated in a Galactomannan-Chitosan Composite for the Evaluation of Phenolic Compounds.

Galactomannan, a neutral polysaccharide, was extracted from carob seeds and characterized. It was used for the first time for the fabrication of a laccase-based biosensor by the encapsulation of laccase in a chitosan+galactomannan composite. The fabricated biosensor was characterized by FTIR, scanning electron microscopy and cyclic voltammetry. The pyrocatechol detection was obtained by cyclic voltammetry measurements, through the detection of o-quinone at −0.447 V. The laccase activity was well preserved in the chitosan+galactomannan composite and the sensitivity of detection of pyrocatechol in the 10−16 M–10−4 M range was very high. The voltammetric response of the biosensor was stable for more than two weeks. To estimate the antioxidant capacity of olive oil samples, it was shown that the obtained laccase-based biosensor is a valuable alternative to the colorimetric Folin–Ciocalteu method.

Application of cold plasma corona discharge in preparation of laccase-based biosensors for dopamine determination.

Laccase-based biosensors were successfully prepared using innovative, cheap, one-step Soft Plasma Polymerization technique by deposition of a bio-recognition layer on glassy carbon electrode and MWCNT (Multi-walled Carbon Nanotubes)-modified glassy carbon electrode. The Soft Plasma Polymerization technique is based on corona discharge of cold atmospheric plasma with close to room temperature. The presented work includes study of biosensor working conditions, optimization of the voltage value applied for corona discharge generation as well as applicability and interference studies for dopamine determination. The biosensor constructed under optimal conditions (corona discharge generated at a voltage of 3kV and in 30s time deposition, helium flow rate 10L/min, laccase solution flow rate 200μL/min) has two linear ranges from 0.1μmol/dm3 to 10μmol/dm3 and from 10μmol/dm3 to 50μmol/dm3 with dopamine detection sensitivities of 3.63μA*dm3/μmol and 1.33μA*dm3/μmol. Application of the MWCNT interlayer allows the dopamine detection sensitivity to be significantly increased to 22.35μA* dm3/μmol for a linear range from 0.1μmol/dm3 to 6μmol/dm3. Additionally, the studied biosensors have stable and anti-interference ability. Both biosensors were successfully applied for dopamine determination in pharmaceutical preparation.

Electrospray deposition as a smart technique for laccase immobilisation on carbon black-nanomodified screen-printed electrodes

Enzymes immobilisation represents a critical issue in the design of biosensors to achieve standardization as well as suitable analytical performances in terms of sensitivity, selectivity, and stability. In this work electrospray deposition (ESD) has been exploited as a novel technique for the immobilisation of laccase enzyme on carbon black modified screen-printed electrodes. The aim is to fabricate an amperometric biosensor for phenolic compound detection. The electrodes produced by ESD have been analysed by scanning electron microscopy and characterised electrochemically to prove that this immobilisation technique is suited to manufacture high performance biosensors. The results show that the laccase enzyme maintains its activity after undergoing the electrospray ionisation process and deposition and the fabricated biosensor has improved performances in terms of storage (up to 3 months at room temperature) and working (up to 25 measurements on the same electrode) stability. The laccase-based biosensor has been tested for phenolic compound detection, with catechol as target analyte, in the linear range 2.5–50 μM, with 2.0 μM limit of detection, without interference from lead, cadmium, atrazine, and paraoxon, and without matrix effect in drinking, surface, and wastewater.

Covalent attachment of laccase to carboxymethyl-botryosphaeran in aqueous solution for the construction of a voltammetric biosensor to quantify quercetin

Laccase from Botryosphaeria rhodina MAMB-05 was covalently immobilized on carboxymethyl-botryosphaeran by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) in aqueous solution. This approach was employed to fabricate a novel laccase-based biosensor to electrochemically quantify quercetin (QCT), using a simple carbon black paste electrode as a transducer. The proposed biosensor was characterized by electrochemical impedance spectroscopy and Nyquist plots were used to evaluate the immobilization of the enzyme. For determining QCT, variables were optimized, that included experimental conditions for laccase immobilization, pH of the supporting electrolyte, and instrumental parameters of the electroanalytical technique. From square-wave-voltammograms, a linear dependence between the cathodic current peak and QCT concentration was observed within the range 4.98–50.0 × 10−8 mol L−1, with a theoretical detection limit of 2.6 × 10−8 mol L−1. The proposed method was successfully applied to determine QCT in beverages, pharmaceuticals, and biological samples. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in chemically complex solutions. Operational and analytical stability of the laccase-biosensor were evaluated, and good intra-day (SD = 1.23%) and inter-day (SD = 2.32%) repeatability, and long storage stability (SD = 3.47%) are presented.

Chemometric-assisted construction of a biosensing device to measure chlorogenic acid content in brewed coffee beverages to discriminate quality

In this work we propose the use of statistical mixture design in the construction of a biosensor device based on graphite oxide, platinum nanoparticles and biomaterials obtained from Botryosphaeria rhodina MAMB-05. The biosensor was characterized by electrochemical impedance spectroscopy. Under optimized experimental parameters by factorial design, the biosensor was applied to the voltammetric determination of chlorogenic acid (CGA) measured as 5-O-caffeoylquinic acid (5-CQA). The biosensor response was linear (R2 = 0.998) for 5-CQA in the concentration range 0.56–7.3 µmol L−1, with limit of detection and quantification of 0.18 and 0.59 µmol L−1, respectively. The new biosensing device was applied to quality control analysis based upon the determination of CGA content in specialty and traditional coffee beverages. The results indicated that specialty coffee had a significantly higher content of CGA. Principal component analysis of the voltammetric fingerprint of brewed coffees revealed that the laccase-based biosensor can be used for their discrimination.

New electrochemical laccase-based biosensor for dihydroxybenzene isomers determination in real water samples

The toxicity of catechol, hydroquinone and resorcinol requires their determination at low concentration levels over a wide linear range. Therefore, in this work we present a new bio-sensing device fabricated by a Soft Plasma Polymerization (SPP) technique for the determination of dihydroxybenzene isomers in real water samples. The Corona SPP technique was applied to the deposition of a bio-recognition layer based on the laccase enzyme. In comparison to conventional methods of biosensor construction, application of the SPP technique allows reduction of its preparation time from several hours to ∼2 min with no chemical consumption. The fabricated biosensor was characterized to find the optimum experimental conditions and determine its response time, sensitivity, linear range and selectivity coefficient. The optimum environment of the bio-sensor for generation of catechol, hydroquinone and resorcinol oxidation peaks was found to be acetate buffer at pH = 6.13. The biosensor exhibited a wide linear response both to catechol, hydroquinone and resorcinol with good sensitivity and high regression coefficient. Further studies showed also that the fabricated biosensor can be successfully applied to simultaneous determination of hydroquinone and resorcinol as well as catechol and resorcinol.

Promoting laccase sensing activity for catechol detection using LBL assemblies of chitosan/ionic liquid/phthalocyanine as immobilization surfaces

The performance of electrochemical laccase-based biosensors can be improved by immobilizing the enzyme on composite Layer-by-Layer (LbL) supports in which materials with complementary functions are combined. LbL films are formed by layers combining an electrocatalytic material which favors electron transfer (sulfonated copper phthalocyanine, CuPcS(−)), an ionic liquid which enhances the electrical conductivity of the layers (1-butyl-3-methylimidazolium tetrafluoroborate, IL(+)) and a material able to promote enzyme immobilization (chitosan, CHI(+)). Composite films with different structures have been demonstrated to be efficient electrocatalysts, producing an increase in the magnitude of the responses towards catechol. The most intense and reproducible electrocatalytic effect was observed when a layer of the CuPcS(−) was placed on top of a layer formed by a mixture of CHI(+) + IL(+) to obtain [CHI(+) + IL(+)|CuPcS(−)]2 films.Biosensors with laccase immobilized on the surface of the LbL layers [CHI(+) + IL(+)|CuPcS(−)]2|Lac showed mediated electron transfer between the redox enzyme and the film and a reproducibility of device-to-device performance of 4.1%. The amperometric biosensor showed a sensitivity of 0.237 A·M−1 and a linear detection range from 2.4 μM to 26 μM for catechol. The excellent Limit of detection (LOD) of 8.96·10−10 M (3·σ /m) is one order of magnitude lower than that obtained in similar studies. A Michaelis-Menten constant of 3.16 μM confirms excellent enzyme-substrate affinity.

Dependence of operational parameters of laccase-based biosensors on structure of photocross-linked polymers as holding matrixes

Laccase-based amperometric enzyme biosensors of the third generation for analysis of phenol derivates have been constructed using graphite rods (type RW001) as working electrodes and the photocross-linked polymers as a matrix. Such matrix consisted of epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD) as reactive diluent and 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI) as photoinitiator. The synthesis was made by the reaction of ELO and 10 mol.% or 30 mol.% of RD, using 3 mol.% of PI (ELO/10RD and ELO/30RD, respectively). The holding matrixes were used for an immobilization of commercial laccase from the fungus Trametes versicolor. The network properties of the polymer matrixes, holding biosensing element, were studied by means of swelling and positron annihilation lifetime spectroscopy measurements. The amperometric enzyme biosensor parameters were evaluated using cyclic voltamperometry and chronoamperometric analysis. A correlation between the constructed biosensor parameters and microscopical free volume of the biosensor holding matrixes was established.

Electrosynthesis of polydopamine films - tailored matrices for laccase-based biosensors

This work explores the electrosynthesis of bio-inspired polydopamine thin films (ePDA), a route that is still overlooked in polycatecholamine coatings formation, aiming at the construction of prime matrices for biosensor applications. A combination of surface characterization techniques attests the electrochemical and optical properties, wettability, morphology, thickness and chemical composition of the ePDA films. ePDA coatings exhibit structural differences from the chemically synthesized polymers unveiling distinct polymerization pathways. We propose that fast electropolymerization forms a co-polymer enriched by chains of dopamine in the open form, in detriment of indole moieties, yielding more organized and conducting polymeric matrices. For thin coatings (ca. 3 nm), ePDA presents high electroactivity of pendent quinone groups, appropriate for covalent biomolecule interaction. The suitability of the functional ePDA films towards electrochemical biosensing was demonstrated through the catalytic activity of immobilized laccase. A clear improvement of transducing performances towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) detection was achieved when using the more electroactive ePDA thin film as supporting matrix, reaching a sensitivity of 342 mA M−1 cm−2. The new structural insights presented in this work, undoubtedly prove the advantage of electrosynthesis regarding molecular oxygen-driven polymerization to prepare more reproducible and organized polycatecholamines for amperometric sensors.

Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects.

Laccases exhibit a wide range of applications, especially in the electrochemical field, where they are regarded as a potential biotic component. Laccase-based biosensors have immense practical applications in the food, environmental, and medical fields. The application of laccases as biocathodes in enzymatic biofuel cells has promising potential in the preparation of implantable equipment. Extensive studies have been directed towards the potential role of fungal laccases as biotic components of electrochemical equipment. In contrast, the potential of prokaryotic laccases in electrochemistry has been not fully understood. However, there has been recent and rapid progress in the discovery and characterization of new types of prokaryotic laccases. In this review, we have comprehensively discussed the application of different sources of laccases as a biocatalytic component in various fields of application. Further, we described the potential of different types of laccases in bioelectrochemical applications.

Electroanalysis and laccase-based biosensor on the determination of phenolic content and antioxidant power of honey samples

Honey is a functional food widely consumed. Thus, the evaluation of honey samples to determine its phenolic content and antioxidant capacity (AOC) is relevant to determine its quality. Usually AOC is performed by spectrophotometric methods, which lacks reproducibility and practicality. In this context, the electroanalytical methods offer higher simplicity and accuracy. Hence, the aim of this work was to use of electroanalytical tools and laccase based biosensor on the evaluation of AOC and total phenol content (TPC) of honey samples from different countries. The antioxidant power established by electrochemical index presented good correlation with the spectrophotometric FRAP (Ferric Reducing Ability of Plasma) and DPPH (2,2-Diphenyl-1-Picrylhydrazyl) radical scavenging assays. Also, TPC results obtained by the biosensor agreed with the Folin-Ciocalteu (FC) assay. In addition to the semi quantitative results, the electroanalysis offered qualitative parameters, which were useful to indicate the nature of major phenolic compounds.