Laccase-based biosensors for detection of phenolic compounds

Monitoring of phenolic compounds in the food industry and for environmental and medical applications has become more relevant in recent years. Conventional methods for detection and quantification of these compounds, such as spectrophotometry and chromatography, are time consuming and expensive. However, laccase biosensors represent a fast method for on-line and in situ monitoring of these compounds. We discuss the main transduction principles. We divide the electrochemical principle into amperometric, voltammetric, potentiometric and conductometric sensors. We divide optical transducers into fluorescence and absorption. The amperometric transducer method is the most widely studied and used for laccase biosensors. Optical biosensors present higher sensitivity than the other biosensors. Laccase production is dominated by a few fungus genera: Trametes, Aspergillus, and Ganoderma. We present an overview of laccase biosensors used for the determination of phenolic compounds in industrial applications. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


Introduction
Monitoring of phenolic compounds in the food industry and for environmental and bio-medical analyses, by using portable, costeffective devices, has become an area of growing interest over the past decade. Phenolic compounds are widespread in nature. They can be found in fruits and vegetables, and they are responsible for the organoleptic properties of some food products, such as wine and olive oil [1,2]. Their antioxidant properties help to prevent cancer and cardiovascular diseases [3]. Phenols are also breakdown products from natural organic compounds, such as humic substances, lignins and tannins. However, some phenols are ubiquitous pollutants that come to natural waters from the effluents of chemical industrial activities, such as coal refineries, pharmaceuticals, production of resins, paints, textiles, petrochemicals, and pulp, including the manufacturing of phenol [4,5].
Consequently, aquatic organisms, including fish, are subjected to these pollutants [5], and, due to their toxicity, some phenolic compounds are subject to regulation as water pollutants. In this context, both the European Commission (EC) and the US Environmental Protection Agency (US EPA) have created lists and classifications to prioritize hazardous substances for their monitoring in drinking or natural waters, and, among them, special attention has been devoted to phenolic compounds {e.g., polycyclic aromatic hydrocarbons (PAHs) and organophosphates [6][7][8]}. Of particular concern are emerging pollutants, mainly those occurring in phenolic compounds, with endocrine-disrupting activity, and those represented by chlorophenols and their derivatives [9,10].
Endocrine-disrupting chemicals are substances that mimic the effects of hormones [11], producing adverse effects on reproduction in wildlife and humans; some phenolic compounds, such as bisphenol A, nonylphenol and their alkylphenolic derivatives, triclosan, genistein and others, widely used in industrial and domestic applications, produce estrogenic activity [10,12]. Chlorophenols too are harmful substances, widely used as bleaching agents in the textile and paper industries, and as pesticides in agriculture [13]. Among chlorophenols, 2,4-diclorophenol and 2,4,6trichlorophenol are chemicals produced in large quantities, and are listed as priority environmental pollutants by the US EPA [7].
Although spectrophotometric and chromatographic techniques are the most common methods for the determination of phenolic compounds, capable of identifying and quantifying them with great accuracy, a wide variety of pollutants exist at trace levels [14], and the most recent research in monitoring techniques is mainly focused on bioanalytical tools, such as biosensors, which offer advantages over classical analytical techniques in terms of selectivity, sensitivity, short assay times, and reduced cost of analysis [14].
The use of enzymatic biosensors has increased over time, due to their specific and peculiar properties. Enzymes are versatile, efficient and specific catalysts acting in all chemical reactions that occur under mild conditions [15]. Some biosensor research has been carried out on the detection of phenolic compounds based on enzymes, such as tyrosinase [16,17] and horseradish peroxidase [18]. Nevertheless both these enzymes present some disadvantages: • tyrosinase suffers from low stability and significant inhibition by reaction products; • while horseradish peroxidase needs the presence of hydrogen peroxide to complete its catalytic function [19].
However, laccase appears a strong candidate as a biosensor, providing some specific advantages over other enzymes, such as its ability to catalyze electron-transfer reactions without additional cofactors, oxidize phenols and o,m,p-benzenediol compounds in the presence of molecular oxygen, and good stability [20].
The current review presents an overview of laccase biosensors used for the determination of phenolic compounds in important areas, such as clinical, bio-medical, environmental contamination, industrial and pharmaceutical analysis.

Laccases
The enzyme laccase (polyphenoloxidase; EC 1.10.3.2) is a member of the blue multi-copper-oxidase family. These enzymes have been studied for a long time, due to their ability to oxidize a variety of organic substrates, and to reduce molecular oxygen to water [21]. These enzymes have been detected in a variety of organisms, such as bacteria, fungi, plants, and insects, mainly as extracellular enzymes, although intracellular laccases have also been detected [22]. In plants, laccases are found in the xylem, where they presumably oxidize monolignols in the early stages of lignification, and also participate in the radical-based mechanisms of lignin polymer formation, whereas fungal laccases play a variety of roles, such as morphogenesis, pathogenesis, and lignin degradation [23]. Their unique characteristics have been widely studied and extended to several uses in agricultural, industrial, medical and environmental applications [23,24].

Laccase characteristics and catalytic mechanism
In general, a fungal laccase has a molecular mass of 60-80 kDa and an isoelectric point of 4-7, depending on glycosylation. Laccase is made up of a cluster of four copper atoms (type I copper; type II copper and two type III copper atoms) that form the active site of the enzyme [28]. These copper atoms are classified into three groups, depending on the characteristics obtained by UV/visible and electron paramagnetic resonance (EPR) spectroscopy: • type I copper (T1, ligated by at least one Cys and two His) is responsible for the intense blue color of the enzyme, has a strong electronic absorption at~600 nm and is EPR detectable; • type II copper (T2, ligated by two His) shows no absorption in the visible spectrum (colorless) but reveals detectable EPR properties; and, • type III copper consists of a pair of anti-ferromagnetically coupled copper atoms (T3, each ligated by three His) spectroscopically characterized by a weak adsorption at 330 nm (oxidized form) and by the absence of an EPR signal [26,29].
Upon strong anion binding (e.g., azide and fluoride), perturbation on the EPR spectra of laccase is observed, causing disruption of the anti-ferromagnetic coupling of T3 and in the T2 EPR signal intensity [30,31]. The T2 and T3 copper atoms form a trinuclear cluster where reduction of molecular oxygen and release of water takes place [26,29].
Laccases have activity toward ortho-and para-diphenol groups, including mono-, di-, and poly-phenols, aminophenols, methoxyphenols, aromatic amines and ascorbate, with the concomitant four-electron reduction of oxygen to water [26,32]. The catalytic mechanism of the laccase starts with the donation of an electron to the substrate by the T1 copper site, followed by an internal electron transfer from the reduced T1 to the T2 and T3 copper sites. The T3 copper functions as a two-electron acceptor in the aerobic oxidation process, in which the presence of the T2 copper is necessary. The reduction of oxygen to water takes place at the T2 and T3 cluster and passes through a peroxide intermediate [29,33] (see Fig. 1).
Laccases are unable to oxidize directly non-phenolic substrates or large molecules with a high redox potential [34]. In this context, laccase-mediator compounds act as intermediate substrates for laccase, producing a high redox potential intermediate able to oxidize indirectly non-phenolic substrates, increasing the range of laccaseoxidizable compounds [24,26].
Being an oxidoreductase, the laccase has Cu sites with defined redox potentials (E 0 ). For the T1 and T3 Cu, some laccases have a "low" E 0 of ≈0.4-0.5 V (versus the normal hydrogen electrode), while others have a "high" E 0 of ≈0.7-0.8 V. However, the E 0 of the T2 Cu appears to be ≈0.4 V for both low and high E 0 laccase groups [35]. In general, a bell-shaped pH-activity profile (optimal pH) at ≈5-7 is observed for phenols, anilines or other substrates whose oxidation by laccase is accompanied by H + . Because of the oxidative H + release, the E 0 of these substrates decreases as pH increases [35]. The subsequent increase of the ΔE 0 with laccase enhances enzymatic oxidation, contributing to the ascending part of the pH profile. However, at higher pH, the laccase inhibition by OHbecomes more pronounced, contributing to the eventual descent of the pH profile.

Application
The ability of laccases to oxidize a broad range of phenolic compounds employed in several industrial sectors has increased their biotechnological potential. The most common uses of laccase are in: • the textile, pulp and paper industries for bleaching and breaking down lignin [24]; • wastewater treatment for dye decolorization and xenobiotics degradation [36,37]; • the food industry [24]; and, • the development of biofuel cells [38,39].
Laccases have also been used in the design of biosensors for the detection of phenolic compounds in food [1,40,41], and for environmental [2,42] and medical applications [43,44].

Immobilization of laccases in biosensors
To become viable industrial catalysts, laccases need to be subject to treatments in order to make them robust, recyclable, or heterogeneous. One of the most studied treatments is immobilization, defined as attachment of an enzyme to an insoluble support [45]. It is achieved by chemical linkage or physical adsorption/entrapment in carriers [46]. The benefits of an efficient protocol of immobilization are very important, namely prolonged use of the sensor and anticipated extended storage and working stability [47].
The ideal support should be inert, stable and resistant to mechanical forces. It is also important to consider shape, distribution and pore size and expandability. Stability, selectivity and activity of an enzyme are obtained by combining immobilization techniques with proper selection of the support [48]. For example, the method by which an enzyme is immobilized at an electrode surface is a critical factor to establish efficient electron transference between the enzyme and the electrode surface [49]. Furthermore, covalent coupling to a solid carrier may give the enzyme increased resilience against pH or thermal inactivation, although the immobilization may lead to a significant loss in activity of the original enzyme [46].

Immobilization methods
The selection of the immobilization method depends on the nature of the biological element and its application. For biosensing applications, it is necessary to take into account the type of transducer used, the physicochemical properties of the analyte and the operating conditions in which the biosensor is to function [50]; all these considerations will allow the biological element to exhibit maximum activity and help the stability and the reusability of the device. Common methods of laccase immobilization in biosensors are covalent binding, adsorption, cross-linking, encapsulation and entrapment (see Fig. 2) [48].
Covalent binding is based on the chemical activation of groups in the support matrix so that they react with functional groups in the biomaterial, which are not essential to catalytic activity. This method presents high stability and allows the enzyme to have some resistance to the effects of temperature, pH and other conditions; however, possible changes in the structure of the active center can occur [51]. Adsorption is a simple, low cost and fast immobilization method. The enzyme is bound to a support via ionic interactions or weak forces, such as van der Waals and hydrogen bridges. However, the biological elements immobilized through this method are mechanically unstable and can be easily desorbed under operating conditions [52].
Cross-linking bonding uses bifunctional reactives to generate intramolecular bonds between the molecules of the enzyme. These cross-linkers include dialdehydes, diiminoesters, diisocyanates and diamines activated by carbodiimide [51]. Under this type of immobilization, the complex-enzyme formed is resistant to extreme conditions of pH and temperature. However, large quantities of enzyme are needed, and factors, such as pH and ionic strength, must be controlled [53].
In encapsulation, the bioactive agent is confined to the core of micron-sized spheres made from a semi-permeable material, such as polymers [54]. Entrapment refers to a mixture of the biomaterial with a monomer solution that is then polymerized to a gel, trapping the biomaterial within the interstitial spaces of the polymeric gel. It can also be occluded within the microcavities of a synthetic fiber [55]. The enzyme suffers minimum alteration, but the method has some disadvantages, such as continuous leakage of enzyme due to variable pore-size, uneven distribution in the gel and reduced substrate accessibility to the enzyme by diffusional limitation [48]. Table 1 shows examples of immobilized laccase systems in biosensing applications.

Table 1
Methods of laccase immobilization reported by support and the immobilization characteristics of the solid biocatalyst Ref.
Immobilization/material Support Comments Method of immobilization: Absorption [56] Carbon nanotubes (CNTs) in surface of modified glassy-carbon electrode -Good mechanical and electronic properties -Biocompatibility with a variety of enzymes -Ability to facilitate electron transfer -In aqueous solution, poor solubility of CNTs, which need some organic solvent to solubilize Method of immobilization: Covalent binding of the enzyme and cross-linking with glutaraldehyde [57] N-succinimidyl-3-thiopropionate (NSTP) in Gold electrodes -With the cross-linking immobilization procedure a larger amount of enzyme was present on the electrode surface, much more sensitive than the covalent binding -Cross-linking three-dimensional network plays an important role in substrate accessibility to active centers of the enzyme Method of immobilization: Covalent attachment of the enzyme in cellulose acetate support modified with ionic liquid [58] Carboxymethyl cellulose with the ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide /silica or a polymer -Immobilization of more active forms of the species with the establishment of a porous contact region between the phases within the structure -Highly efficient and robust biocatalysts

Transduction principles in laccase biosensors
A biosensor is an analytical device, which converts into an electric or other kind of signal, the physical or chemical properties of a biological system (e.g., enzymes, antibodies, receptors, cells, or microorganisms), which is in direct contact with the sample. The amplitude of the signal depends on the concentration of defined analytes in the solution [59].
A biosensor (Fig. 3) has two basic components: • the bioreceptor (biochemical-recognition system), which translates information from the biochemical domain into a chemical or physical output; and, • a transducer, which converts the output chemical signal of the recognition system to an electrical domain [60].
Enzymes were historically the first molecular recognition elements used in biosensors, and continue to be the basis of a significant amount of published research, due to the relative simplicity of modifying their catalytic properties or substrate specificity by genetic engineering. Catalytic amplification can also be achieved by modulating the enzyme activity with respect to the target analyte [61].
In this context, laccase appears a strong candidate for biosensing applications, providing some specific advantages over other enzymes: • the ability to catalyze electron-transfer reactions without additional cofactors; • the ability to oxidize phenols and o,m,p-benzenediols in the presence of molecular oxygen; and, • good stability [20].

Electrochemical biosensors
Electrochemical biosensors are normally based on the production of electrons by enzymatic catalysis. The target analyte is involved in the reaction, which takes place on the active electrode surface, and the ions produced create a potential, which is subtracted from that of the reference electrode to give a measurable signal [14,62]. Depending on the property to be measured by the detector system, electrochemical biosensors may be divided into conductometric, potentiometric and voltammetric/amperometric biosensors.
The ability of laccases to catalyze the oxidation of phenolic and non-phenolic compounds, coupled with the reduction of molecular oxygen to water, makes them valuable for commercial applications [35]. Characteristics, such as good mechanical and electronic properties, and their ability to transfer electrons make laccases good candidates to immobilize biomolecules. The surface of the electrodes allows stability and biocompatibility and promotes development and application of these techniques in the electrochemical field.
Conductometric biosensors measure changes in the conductance between a pair of metal electrodes as a consequence of the activity of a biological component; many enzyme reactions may be monitored by ion conductometric or impedimetric devices, using interdigitated microelectrodes [62].
Potentiometric measurements involve determination of the potential difference between an indicator and a reference electrode, or two reference electrodes separated by a selective membrane [62].
Voltammetric/amperometric biosensors measure the changes in the current on the working electrode due to direct oxidation of the products of a biochemical reaction [63].

Voltammetric sensors
Voltammetry in general is the measurement of the current that flows through an electrode as a function of the potential applied to it, and the result is a current/potential curve [64].
Fernandes et al. [65], described the construction of a biosensor based on laccase immobilized on microspheres of chitosan crosslinked with tripolyphosphate by spray drying. It is being used for rutin determination in pharmaceutical formulations, obtaining a bioelectrode that exhibits high sensitivity, good reproducibility, low detection threshold and rapid response. Santhiago et al. [66] designed a biosensor based on a carbon-paste electrode modified with laccase, produced by Aspergillus oryzae for the determination of l-cysteine in pharmaceutical formulations, performed in the presence of hydroquinone. Gupta et al. [67] used Coriolus hirsutus laccase immobilized onto amine-terminated thiol monolayers on a gold electrode to monitor catechol .
The use of biosensors based on nanocomposites is widely studied. Chen et al. [49] used zein, a natural biodegradable protein polymer, to design a new composite of laccase-gold nanoparticles (AuNPs)cross-linked zein ultrafine fibers (CZUFs) for the determination of catechol. The results demonstrated that this biosensor possessed a high detection sensitivity, which was attributed to direct electron transfer (DET) [49].
Ionic liquids (ILs) have attracted considerable attention for electroanalysis because of their unusual physical and chemical properties, mainly resulting from their peculiar structural organization. Of particular interest are salts resulting from the combination of imidazolium cations with inorganic or organic anions that are liquid at room temperature [68]. They are widely used in laccase biosensors to prepare modified electrodes and in preparation and stabilization of nanomaterials, such as platinum NPs (PtNPs) and AuNPs dispersed in ILs [69][70][71]. Table 2 shows a comparison between voltammetric laccase biosensors.

Amperometric laccase biosensors
Amperometric sensors are a special classification of voltammetric sensors, where potential is kept constant as a function of time. The current generated by oxidation or reduction of redox species at the electrode surface, which is maintained at an appropriate electrical potential, is measured [62]. The current observed has a linear relationship with the concentration of the electroactive species. The electrode is usually constructed of platinum, gold or carbon. Adjacent to the electrode, entrapped by a membrane or directly immobilized, is the enzyme involved [14].
Recently, there was increasing focus on the use of nanomaterials, leading to the improvement of the analytical performance of enzyme electrodes. Timur et al. [78] developed a thick film sensor immobilizing laccases from different sources in a polyaniline (PANI) matrix; a conducting polymer with high conductivity, chemical durability and good environmental stability [78,79] for the determination of phenolic compounds. The measurement of oxygen consumption is related to the oxidation of the analyte.
Chawla et al. [63] described the construction of a biosensing platform for the determination of total phenolic content in fruit juices by fabricating nickel NPs (NiNPs) covered with carboxylated multiwalled-carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold electrode and modified with laccase. This nano-composite-modified electrode combines the ability of CNTs and a conductive polymer to promote electron-transfer reactions with the advantages of entrapping biological material [80].
Rahman et al. [81] tested Den-AuNP nanocomposites to immobilize laccase (PDATT/ Den(AuNPs)/laccase) covalently to fabricate a third-generation catechin biosensor. The biosensor developed in this study is a promising tool for the detection of catechin in food and biological samples, combining physical and chemical properties of AuNPs and the surface reactivity of dendrimers.
Vianello et al. [82] presented a flow biosensor based on a monomolecular layer of laccase from Rigidoporus lignosus immobilized on a gold support. This biosensor detects phenols in the low micromolar range [i.e., below the European Community limits (0.5 mg/L)] [82].
Kulys et al. [83] employed, for the first time, recombinant fungal laccase from Polyporus pinsitus and thermostable recombinant laccase from Myceliophthora thermophila for biosensors using printed graphite electrodes suitable for continuous flow-through measurements of phenolic compounds in alarm systems.
In the food industry, to ensure the good quality of the final products, it is necessary to measure some attributes at different stages of the production process. For this reason, the development of novel analytical sensors in the food industry is needed. The determination of polyphenols in food, employing laccase biosensors, has been widely studied {e.g., in tea leaves at different stages of tea production [41] and commercial fruit juices [80]}.
There are very few papers that describe the application of laccase with the simultaneous use of other enzymes to obtain bi-enzymatic biosensors for the determination of phenolic compounds. Bauer et al. [84] developed an enzyme sensor to measure morphine based on laccase (Lac) and PQQ-dependent glucose dehydrogenase (GDH) immobilized on a Clark oxygen electrode. Laccase oxidizes morphine and the Clark electrode indicates the consumption of oxygen, which is then regenerated by glucose dehydrogenase, enhancing the assay sensitivity. Then, with the objective of discriminating between morphine and codeine in pharmaceutical drugs, a double detector was developed, using the morphine dehydrogenase (MDH)/salicylate dehydrogenase (SHL) -and the LACC/GDH sensors [84].
Tang, et al. [85] used a bi-enzyme horseradish peroxidase/ laccase system for rapid, sensitive detection of E. coli density. Since the E. coli metabolism of salicylic acid (SA) forms polyphenolic compounds, the amount depending upon E. coli density, the biosensor was applied to detect E. coli density through oxidation of the polyphenols in the presence of laccase/HRP. Table 3 shows a comparison between amperometric laccase biosensors.

Conductometric and potentiometric sensors
Conductometric detection involves monitoring changes in the electrical conductivity of the sample solution. As the composition of the medium changes in the course of the enzymatic reaction, whose charged products result in changes in ionic strength, the conductivity increases [120].
In the case of potentiometric sensors, the measuring principle is based on the Nernst equation through measurement of the potential between non-polarized electrodes (working electrode and Linear range 0.990-11.5 μm Food analysis Vegetables [73] (continued on next page)   (continued on next page) (continued on next page)    (continued on next page)   (continued on next page)  [111] (continued on next page) 35  [115] (continued on next page)  reference electrode) [62]. The potentiometric sensors use an electrochemical cell with two reference electrodes to measure the potential across a membrane that selectively reacts with the charged ion of interest. These chemical sensors can be turned into biosensors by coating them with a biological element, such as an enzyme that catalyzes a reaction to produce the ion that the underlying electrode is designed to detect [120]. Since the reactions catalyzed by laccases proceed by the monoelectronic oxidation of a suitable substrate molecule (phenols and aromatic or aliphatic amines) to the corresponding reactive radical (a free cation) [121], the use of laccases would be suitable for the design of biosensors employing both transduction principles. However, a comprehensive search of publications about laccase biosensors using conductometric and potentiometric principles showed a lack of research in this field, making it a target area for future research.

Optical biosensors
During the enzymatic reaction of laccase enzymes, intermediates appear, with different spectroscopic properties [122]. Sanz et al. [123] followed the enzymatic reaction of laccase with phenol using these intrinsic spectroscopic properties, (molecular absorption and fluorescence) using a lac-polyacrylamide sensor film. The results observed in the study show that the lifespan of the sensor film does not depend on the storage time (stored for at least 6 months) but only on the number of measurements performed by the device [123].
The ability of laccase to oxidize methoxyphenols in the presence of 3-methyl-2 benzothiazolinonehydrazone (MBTH) to produce azo-dye compounds was studied by Setti et al. [124]. Based on this concept, Abdullah, et al. [124] developed an optical biosensor using stacked films of MBTH in hybrid Nafion/sol-gel silicate and laccase in chitosan for the detection of phenolic compounds. The same study also demonstrated that laccase immobilization in this hybrid material enabled the biosensor to be more selective to catechol, as compared with another analytes, such as guaiacol, o-cresol and m-cresol that were also tested.
Huang et al. [125] fabricated a fiber-optic biosensor for the determination of adrenaline. They immobilized laccase on CuTAPc-Fe3O4-NPs to catalyze the oxidation of adrenaline and to detect the consumption of oxygen with a fluorescent oxygen-sensing membrane. The adrenaline oxidation leads to a fluorescence change because the molecular oxygen acts as a dynamic quencher of fluorescence [126]. Ferreira et al. [44] combined the HPLC technique with an optic-fiber detection system using laccase as a sensing bioelement (HPLC-LacOF) for quantification of catecholamines (epinephrine, dopamine and norepinephrine) in biological fluids (i.e., plasma and urine). The system was based on changes in the refractive index of the optic-fibersensitive cladding (laccase + alginate matrix) caused by the linkage of the catecholamines eluted from the HPLC.
Zhang, et al. [126] developed a method based on the characterization of a luminol−H2O2−laccase reaction for the detection of E. coli O157:H7, using the ability of laccase to oxidize in a strong alkaline medium, which was compatible with the luminol system. Table 4 summarizes laccase biosensors that employ optical principles for the detection of phenols.

Thermal sensors
Calorimetric or thermal sensors work by determining the presence or the concentration of a chemical species by measuring its enthalpy change [129].
method. The enzyme reactions were monitored by changes in the enthalpy of the reaction system. A comprehensive search of publications on laccase biosensors using this transduction principle found very little research in this field, making it a target for future work. Table 5 shows a comparative analysis of the transduction methods used in laccase biosensors regarding the limit of detection (LOD), stability, response time and lifespan of the device. In the case of electrochemical biosensors, we notice that amperometric principles are widely used in design, using mainly laccase sourced from Trametes, Aspergillus and Ganoderma genera.

Comparative analysis of laccase biosensors
The best lifespan was obtained by electrochemical devices, with stability of up to 10 months and at least 900 measurements.
The biosensor using a strain from Ganoderma lucidium showed a lifespan of 4 months with stability of up to 200 measurements without loss of activity.
Although electrochemical sensors have the advantages of quick response, cost efficiency and simplified operation, they also have disadvantages, such as degradation of the electrode surface by the continuous flow, leading to a lower lifespan and stability, in contrast with optical biosensors, which achieved lower LODs and longer stability.
Optical biosensors, in turn, show other disadvantages, such as requiring multi-step assays, and often large and expensive equipment, such as fluorescence, that requires labeled molecules. In this context, Ferreira et al. [44] developed an optical sensor based on Trametes versicolor immobilized onto an optical fiber to detect catecholamines, which maintained its stability for a period of two months of continuous operation and showed the best LOD, detecting trace levels (pg/mL).

Global analysis of laccase biosensors by research field
We conducted a literature review on the status and progress of research on laccase biosensor design during the period 1992-2014. The methodology was based on a bibliometric analysis, previously described elsewhere [130]. The bibliometric methodologies referred to enumeration and statistical analysis of scientific output in the form of articles, publications, citations, patents and other indicators [131].
Our literature research was based on the following: the transduction principle used the source of laccase and the immobilization technique applied. This study used the number of Web publications in the Scopus database as a reference, as has been done for other studies from different areas of knowledge such as chemical engineering, environmental sciences, separation and purification technologies [132][133][134][135][136][137][138][139][140][141]. Scopus was used as the database of choice, since it covers most of the journals included in the Thomson Reuters Web of Science [142].
We found that the electrochemical principle was the most studied method for biosensor design with laccases ( Fig. 4), where 153 documents have been published. Since 2001, scientific production in this field has grown considerably. By comparison, optical and thermal biosensors using laccases received relatively little attention, with only 13 documents each. However, a deeper look into thermal research published found that this principle is only mentioned, but has not been developed. Fig. 5 shows the main genera of organisms used as laccase source for biosensor design during the period 1992-2014 worldwide. Of the sources of laccase, the genus Aspergillus is the most widely used in biosensors, contributing to about 53% of laccase followed by Trametes (14%) Ganoderma (9%), Coriolus (8%), Pleurotus (6%), Pycnoporus (6%) and finally Rhus (4%). We note that more research needs to be developed for Rhus, Pycnoporus and Pleurotus as laccaseproduction organisms for biosensors.

Barriers and solutions for laccase biosensors
Laccase biosensors show great potential for use in the food industry, environmental monitoring and biomedical analysis. Despite  the advantages of laccase biosensors, some problems need to be addressed in order to generalize their use in industrial processes.
Since immobilization, one of the main steps in biosensor fabrication, might reduce enzymatic activity, other technologies could be coupled to counteract this effect. Incorporation of nanomaterials has been demonstrated to improve sensitivity and overall performance of enzymatic biosensors, especially with AuNPs, CNTs and graphene [143].
Although the electrochemical principle is the most studied in terms of laccase biosensors, there is little research regarding conductometric and potentiometric principles. The lack of research in these transducers is probably due to the instability and short life of the reactive radicals (charged products) produced by laccases, which cannot be detected before they undergo further oxidations and non-enzymatic reactions (e.g., hydration, disproportion or polymerization) [144]. However, amperometry is the transducer most used [145]. Amperometric detection is commonly used with biocatalytic sensors because of its simplicity and advantages, since the fixed potential during amperometric detection results in a negligible charging current (the current needed to apply the potential to the system), which minimizes the background signal that adversely affects the LOD [120]. Another advantage of amperometric detection is the significantly enhanced mass transport to the electrode surface. The high selectivity due to the oxidation or reduction potential used for detection is characteristic of the analyte species [59]. The high costs related to enzyme production, immobilization and biosensor fabrication might reduce its feasibility. Electrochemical sensors, based on silicon, show great potential for batch fabrication and could therefore be best suited for disposable sensors [146].
Electrochemical interference from substances, such as paracetamol, ascorbic acid or uric acid, can disturb an accurate measurement if the biosensor is operated at high applied potentials. This might be overcome by using a laccase-covered electrode and a blank electrode [147].
Enzyme instability, and consequently insufficient performance, are the main problems in microelectronic amperometric devices. Membrane fouling caused by protein adsorption leads to a decrease in sensitivity. A combination of different technologies, such as electropolymerization and photo-patternable enzyme membranes, can lead to reliable biosensor systems [147].

Conclusions
The use of laccase biosensors shows great potential for detection and quantification of phenolic compounds, which are regulated by different directives, such as the European Community Directive, Japan's Ministry of Health and Labor and Welfare, and the US EPA. Conventional detection methods, such as spectrophotometry, gas chromatography, liquid chromatography and capillary electrophoresis, are time consuming and expensive. The main transduction methods for laccase biosensors are electrochemical (amperometric, voltammetric, potentiometric, conductometric), optical and thermal. The most widely studied and used of these methods for laccase biosensors is the amperometric, whereas thermal and conductometric principles are the least studied and applied. Optical biosensors provide a higher sensitivity, which could be used in the food industry or in the biomedical field, where high quality is required.
In order to expand their use for industrial processes, some problems need to be tackled, which could be done by integrating inexpensive supports and immobilization techniques to avoid enzyme leakage, improving the activity recovery during the immobilization process or by incorporating nanomaterials.
Laccase biosensors represent a fast method for monitoring online and in situ phenolic compounds, with high sensitivity and reproducibility, and can be standardized for food, environmental and medical industries.