Optical Properties of Laccases and Their Use for Phenolic Compound Detection and Quantification: A Brief Review

Abstract

Phenolic compounds (PheCs) are particularly relevant in many different frameworks due to their pro-oxidant and antioxidant activities. In fact, on the one hand, they are considered very dangerous pro-oxidant agents that can be present in the environment as pollutants in wastewater and soil from different industrial and agricultural industries. On the other hand, the antioxidant influence of PheCs available in natural products (including foods) is nowadays considered essential for preserving human health. Conventional techniques for detecting PheCs present some disadvantages, such as requiring expensive instrumentation and expert users and not allowing in situ measurements. This is the reason why there is a high interest in the development of simple, sensitive, specific, and accurate sensing methods for PheCs. Enzymes are often used for this purpose, and laccases with unique optical properties are adopted as bio-elements for sensing schemes. The present paper aims to revise the optical properties of laccases and their use for developing PheC detection and quantification methods used in different fields such as environment monitoring, food characterization and medical applications. In particular, the results offered by UV, visible and infrared absorption, fluorescence, Raman, and surface-enhanced Raman spectroscopy (SERS) have been considered. The enzymatic biosensing devices developed using the related optical signals have been reported, and a comparison of their performances has carried out. A brief description of the main characteristics of laccase and phenols is also given.

1. Introduction

Laccases are cuproproteins and are part of a small group of enzymes called polyphenol oxidases or even multicopper blue oxidases [1,2]. In nature, the most relevant role of laccases is related to the polymerization and degradation of lignin, but they are also involved in many other processes as lignification is necessary to maintain the cell wall structure and its mechanical rigidity. Laccases also participate in the feedback responses of plants to environmental stresses, and they contribute to their defense mechanisms. They contribute to wound healing, iron metabolism, and polymerization of phenolic compounds [3,4].

Laccases catalyze the oxidation reaction of diverse substrates such as phenolic and aromatic molecules (ortho- and para-diphenols, amino phenols, methoxy-phenols, poly-phenols), aliphatic amines, and inorganic cations, resulting in water as a product of reduction in molecular oxygen, representing one of the six classes of enzymes that couple the four-electron reduction of O₂ to H₂O with four one-electron oxidations of substrates (Figure 1) [5,6,7].

Laccases have found a very large number of biotechnological applications. The most relevant are wastewater treatment from the textile, paper, and petrochemical industries; herbicides and pesticide bioremediation; decontamination of aquatic and terrestrial environments from phenols; and biosensing. A complete list of laccase applications can be found in [8,9,10]. Among the above-mentioned applications, biosensing represents one of the most investigated in the continuous search of new approaches for developing simple, sensitive, specific, and accurate sensing methods for phenolic compounds. These substances have recently drawn attention given their pro-oxidant and antioxidant effects, which have the potential to impact human health and the environment [11,12].

The electrochemical properties of laccases have already allowed the development of numerous biosensors [13,14,15,16]; conversely, the optical properties of laccases have been used for developing only a limited number of interesting biodevices for applications mainly in food control, environment monitoring and clinics [17]. In fact, reviews summarizing the use of laccase for biosensing applications are mainly focused on electrochemical biosensors, and little attention is devoted to optical biosensing (see, for example, Refs. [7,15,18,19,20,21,22,23,24]. Furthermore, in recent years, the number of new optical biosensors reported in the literature has not been so high, as can be seen by consulting Scopus or other databases. These aspects prompted us to prepare the present review to highlight the potentialities of laccases for advancement in this field; for this purpose, we revised the main relevant results on the optical properties of laccases together with the most significant optical biosensors developed to date. A brief description of the main characteristics of laccase and phenols is also given and, since the laccase immobilization procedures are of fundamental importance in biosensor development, a summary of the most widely used methods is reported in the Supplementary Materials together with a selection of relevant references.

2. Main Laccase Types and Their Structure

Laccase (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductase) is related to the superfamily of multicopper oxidases (MCOs), an enzyme group consisting of many proteins with different substrate specificities and various biological functions. It was one of the first enzymes to ever be identified, an enzyme from the Chinese lacquer tree (Rhus vernicifera) having been first isolated in 1883 [25]. Since then, nearly 140 years of research have disclosed the unique properties and the important roles of these copper-containing enzymes. The first bacterial laccase (Azospirillum lipoferum) was isolated in 1993 from the rhizosphere of rice [26].

To date, about 280 laccases have been characterized according to the BRENDA database (http://www.brenda-enzymes.org/ accessed on 22 September 2023), found mainly in fungi (Basidiomycetous and Ascomycetous) [27] but also in bacteria, insects, and algae [6]. The first bacterial laccase (Azospirillum lipoferum) was isolated in 1993 from the rhizosphere of rice [26]. Nowadays, it is well known that the classification of laccases using only substrate spectra is not accurate because some of them are very similar to those of tyrosinase and bilirubin oxidase [28,29]. A large class of substances (mono-, di-, poly-, and methoxy-phenols, aromatic and aliphatic amines, hydroxyindoles, benzenethiols, carbohydrates, and inorganic/organic metal compounds) can be oxidized by laccases [6,30,31]. Because of this wide substrate range and the fact that laccases use only oxygen as the final electron receptor, they have widespread applications in various industries and in environmental remediation.

Many crystallographic studies on the structure of laccases are now available, especially regarding proteins of fungal or bacterial origin. Plant and animal laccases are difficult to obtain as crystals due to their low abundance and complex purification methods. However, despite their wide taxonomic distribution and substrate diversity, the molecular structure of laccase has proven to be common to all polyvalent copper oxidases [32,33]. Laccases’ domains and amino acid sequences share similarities with those of all multivalent copper oxidases. They have domains like those of copper oxidases and can reduce oxygen to water. The amino acid sequences of laccases, like all multivalent copper oxidases, contain small cupredoxin-like domains of 10–20 kDa and have a relatively simple 3D structure consisting mainly of beta sheets and turns. The crystalline three-dimensional structure of T. versicolor was reported for the first time in 2002 [34]. The molecular mass of monomeric laccase has a molecular mass varying from 60 to 110 kDa with a 10–50% rate of glycosylation. Laccases are thermostable up to 70 °C due to the high carbohydrate content [35].

Fungal laccases are generally expressed in extracellular form, but some fungi can produce intracellular laccases with a molecular weight between 38 and 150 kDa [36]. Laccases typically include three cupredoxin-like domains, like those described in fungi, plants, insects, and some bacteria, mainly consisting of about 500 amino acids.

Bacterial laccases are characterized by a molecular weight between 50 and 70 KDa and are mostly intracellular and belong to two genera, Bacillus and Streptomyces. Small bacterial laccases comprising about 200 amino acids were more recently discovered and considered as two-domain enzymes. Furthermore, similar laccases that are smaller than the typical ones and consist of only two domains can be produced from some fungal species, such as Pleurotus ostreatus [37]. Notwithstanding the low similarity in amino acid sequence between fungal and bacterial laccases, their molecular structure is analogous, and the geometrical spatial arrangement of their active sites is considerably conserved [32,38,39].

Standard laccases contain four copper atoms, which mediate the redox process and are present at three different sites (T1, T2 and T3, defined according to their spectroscopic properties, described in the next section), as shown in Figure 2. The T1 site has a trigonal conformation, with histidine and cysteine acting as equatorial ligands and leucine or phenylalanine acting as axial ligands [40,41]. At the T2 site, copper coordinates with two histidines and a water molecule, while at the T3 site, three histidines and a hydroxyl bridge between the copper pair are present as ligands. The T2 copper center and the two T3 copper atoms form a trinuclear center whose function is to catalyze the reduction of oxygen to water.

Laccases can be grouped considering the redox potential (low, medium, and high) of the T1 site, which influences the efficiency of the enzyme’s catalytic activity [42]. As a rule, a low redox potential (up to 460 mV) is characteristic of bacterial laccases, a medium redox potential (460–710 mV) comprises the laccases of the fungi Ascomycetes and Basidiomycetes, and a high redox potential (more than 710 mV) is typical of the laccases of wood-degrading white rot fungi [43].

Figure 2. Representation of the laccase structure of Trametes versicolor; copper atoms (T1, T2 and T3) are highlighted in brown (reprinted from Ref. [44] under open access conditions).

Figure 2. Representation of the laccase structure of Trametes versicolor; copper atoms (T1, T2 and T3) are highlighted in brown (reprinted from Ref. [44] under open access conditions).

3. Optical Properties of Laccase

3.1. UV–Vis Absorption Spectroscopy

Most laccases have important optical properties due to the presence of copper atoms in the catalytic center, as stated earlier. Most of the kinetic and spectroscopic studies on laccases are summarized in a book by Messerschmidt [45] as well as in several reviews [33,46]. As already mentioned, laccases are also categorized by their optical properties: blue, yellow, and white laccases. Most of the studies reported so far are on blue laccases. In blue laccases, the T1 copper, responsible for the blue color of cuproproteins, has an absorption maximum of about 600 nm [40,47]. These laccases are reported in a variety of organisms, including Pleurotus pulmonarius [48], Pleurotus ostreatus [49,50], Coriolus versicolor, Panus phlebia Radiata, Phlebia tremellosa, Agaricus bisporus [51], Thermus sp. [52], and Trametes versicolor [53]. Those laccases that do not exhibit the absorption feature around 600 nm are known as white or yellow laccases, and they have rarely been studied [54,55]. They differ from blue laccases because they oxidize non-phenolic substrates in the absence of mediator molecules [55], which are required in cases of blue laccases. White laccases have absorption at around 400 nm and contain one copper ion, one iron ion and two zinc ions. They have been observed in Pleurotus ostreatus [49,50], Trametes hirsuta [56,57], and Myrothecium verrucaria [58]. The yellow laccases contain the copper atoms in a modified oxidation state and have been reported in Panus tigrinus [51], Daedalea flavida [59], Trametes sp. [60], Pleurotus ostreatus [61], and Sclerotinia sclerotiorum [54]. As for the copper of type 2, it exhibits weak absorption in the visible spectrum. Finally, the two copper atoms of type 3 are characterized by absorption around 330 nm.

A typical UV–Vis absorption spectrum for a blue laccase has been reported by Shin et al. [62], in their study of the ligninolytic systems of Coriolus hirsutus (a blue laccase) and their role in lignin degradation. The absorption spectrum of laccase showed a signal at approximately 600 nm that is related to T1 copper atoms (Figure 3). The contribution of the copper atoms of type 3 is not clear, but it is associated with a small shoulder that is present at around 330 nm [62].

In 2000, Revina et al. [63] obtained spectroscopic insights into the effect of oxygen in aqueous solutions of two types of laccases: Coriolus hirsutus and Coriolus zonatus. These authors report the optical absorption spectrum of Coriolus hirsutus in a neutral aqueous solution saturated with air in Figure 1 of their paper. In that figure, changes in the 360–600 nm range are evident when the solution is found to be saturated with helium. This phenomenon turns out to be reversible; in fact, after the passage of air, the absorption band returns to the initial state [63]. The same was observed for Coriolus zonatus.

Agrawal et al. [53] investigated the absorption properties of three different types of laccases: blue (Trametes versicolor), white (M. verrucaria ITCC 8447), and yellow (Stropharia sp. ITCC 8422) laccase. Their spectra show significant differences. The absorption spectrum of Trametes versicolor presents a peak at 605 nm (Type I Cu atom), whereas, in the case of white and yellow laccases, the peak is absent and there is a weak absorption band at 400 nm [43].

Shleev et al. [64] presented a work highlighting the optical properties of the laccases from several basidiomycetes (Trametes ochracea, Trametes hirsuta, Coriolopsis fulvocinerea and Cerrena maxima). Spectroscopic investigations were accomplished to understand the characteristics of the active sites of the different laccases mentioned above. In Figure 4, the large absorption band located at 280 nm (typical of tryptophan) is evident, while a small band at 610 nm (related to copper atoms of type 1) is present and more clearly shown in the inset. Also, in this case, a faint shoulder around 330 nm can be devised. Furthermore, Shleev et al. [64] reported the results of their circular dichroism measurements aimed at defining the secondary structures of laccases. These measurements evidenced contributions from α-helix (~10%), β-sheet (~30%), turn (~20%), and random (~40%) components.

Delfino et al. [65] performed a study on the dynamics of the excited state of the T1 copper site of blues laccase from Pleurotus ostreatus, in which they exciting the charge transfer of the 600 nm band with a 15 fs pulse and probing over a broad range in the visible region. The results have been discussed in terms of a peculiar hypothesized trigonal T1 Cu site geometry for the considered enzyme.

The results described here evidence that absorption spectroscopy has been largely used for characterizing different laccases and monitoring their purification processes, but it is also important to consider that the absorption spectral features of laccases are very sensitive to the interaction with the PheCs [42]. In fact, the absorption spectra of the substances produced by the reactions of laccases with different PheCs show different absorption spectral features; for this reason, absorption spectroscopy has been adopted for designing various optical biosensors. Some significant examples of them will be described in Section 5.

3.2. Fluorescence Spectroscopy

Laccases have been observed to possess intrinsic fluorescence that largely depends on tryptophan residues, which can be excited either directly or by energy transfer from tyrosine residues. Golderg and Pecht [66], in a pivotal study on laccase extracted from Rhus vernicifera lacquer, evidenced this behavior by considering the dependence of the emission spectra on the excitation wavelengths. When the excitation wavelength is equal to 305 nm, it is absorbed only by tryptophan, and the maximum of the emission spectra is located at 338 nm. When the excitation wavelength is equal to 280 nm, it is absorbed by tryptophan and tyrosine and the maximum of the emission spectrum shifts to shorter wavelengths with a significant increase in the fluorescence signal intensity. A similar behavior is observed when the excitation wavelength is equal to 250 nm that is absorbed only by tyrosine. In Ref. [66], the changes in quantum yield induced by reduction processes and dependent from excitation wavelength have been also reported.

Using the same laccase cited above, Wynn et al. [67] confirmed that the presence of O₂ influences the reactivity of the enzyme and its excitation and emission spectra. This is because the presence of oxygen influences the oxidation state of the enzyme [67].

The fundamental studies carried out in Refs. [66,67] evidenced the potentiality of fluorescence spectroscopy in characterizing different laccases. Mot et al. [54,68] investigated laccase from the phytopathogenic fungus Sclerotinia sclerotiorum using this spectroscopic approach. The previously cited enzyme presents interesting characteristics; in fact, it can be classified as a yellow laccase by considering its UV–Vis absorption spectral features, but EPR measurements indicate a similarity with blue laccase. In Figure 5, the visible emission spectrum, obtained with an excitation wavelength equal to 330 nm, showing a maximum located at 440 nm, is reported. In this figure, an excitation spectrum with a band at 330 nm is also shown when the fluorescence emission is monitored at 420 nm. This band is a typical characteristic of type-3 copper. Further details on the intriguing optical properties of this enzyme can be found in Refs [54,68].

In the previously mentioned study by Shleev et al. [64], the authors showed the emission and excitation spectra of different blue laccases (see Figure 6). In this case, the excitation spectra clearly show, in almost all of the cases, a band at around 330 nm that is typical of type-3 copper atoms, as above reported. The emission spectra present a maximum of around 440 nm. More recently, the fluorescence properties of laccase from Trametes versicolor were investigated by Saoudi et al. [69]. In particular, the fluorescence emission spectra were collected in the 350–500 nm wavelength region following the excitation of tryptophan residues (λ_exc = 280 nm), and changes induced by different pH levels were also investigated.

Fluorescence spectroscopy has been successfully adopted by several authors for characterizing different aspects of laccases [33,70,71]. In recent years, interest in the application of this technique has particularly been addressed to fungal and bacterial laccases [72,73].

As outlined in Section 5, the fluorescence signal has been adopted for developing a certain number of optical biosensing devices.

3.3. Fourier Transform InfraRed (FT-IR) Spectroscopy

FT-IR spectroscopy is widely employed for investigating the biochemical features of biological samples. The analysis of FT-IR spectra of proteins and enzymes gives valuable information on the functional groups that are present in a particular sample and the changes induced by external agents or occurring processes [74,75,76,77].

For example, FT-IR spectroscopy has been adopted for investigating differences among various laccases. Agrawal et al. [53] acquired spectra from blue (T. versicolor), white (M. verrucaria ITCC 8447) and yellow (Stropharia sp. ITCC 8422) laccases that are reported in Figure 7. These spectra show the main contribution of Amide I in the 1600–1700 cm⁻¹ wavenumber region, Amide II located around 1550 cm⁻¹ and Amide A and B in the 3000–4000 cm⁻¹ spectral interval. As is widely known, the analysis of Amide I allows the characterization of the different subcomponents of secondary structures (mainly α-helix and β-sheet) [74,75]. By performing this analysis, the authors observed that blue laccase shows signals related to the α-helix and β-sheet bands, and prominently those of amides II, A and B (see Figure 7). In the case of yellow and white laccase, the FTIR spectra present similar contributions, but the bands are characterized by different intensities, thus suggesting different relative weights of the various components.

Another relevant application of FT-IR spectroscopy in the case of enzymes is related to the study and characterization of the immobilization processes. In particular, the analysis of the Amide I and Amide II band region is generally used for monitoring the preservation of enzymatic activity after the immobilization procedures, as shown in Refs. [53,78,79,80,81,82] and in further references cited in the Supplementary Materials.

3.4. Raman Spectroscopy

Raman spectroscopy is used to determine the chemical composition of a sample, without any preparation in many cases, and allows information to be obtained from a biochemical point of view without being affected by interference from water molecules.

A study in which the resonant Raman spectra of laccase from Rhus vernicifera in the 350–450 cm⁻¹ region have been obtained by exciting the sample at 647 nm is reported by Musci et al. [83]. Measurements have been made both at room temperature and in a frozen state (200 K). The spectra reported in Figure 8 show the characteristic Raman features of T1 copper sites [84,85,86], which can be ascribed to the mixing of the Cu–S(Cys) stretching vibration with multiple heavy atom bending modes of the ligand and adjacent residues. The intensity of the band at 379 cm⁻¹ is observed to increase when the temperature decreases, while the signal at 403 cm⁻¹ remains unchanged [83].

Laccase from Trametes hirsuta in solution and immobilized in the cubic phase of monoolein have been studied using Raman spectroscopy. Strong bands are observed at 387 and 408 cm⁻¹ along with other bands with weak signals; see Ref. [87].

The resonant Raman spectrum of Pleurotus ostreatus laccase has been reported by Delfino et al. [65], with an excitation of 633 nm in resonance with the T1-absorption feature. The resulting spectrum for the range 300–500 cm⁻¹ is shown in Figure 9. In this case, peaks at 372, 382, 393, 404 and 414 cm⁻¹ together with some shoulders at around 355, 427 and 434 cm⁻¹ can be observed, recalling the characteristic Raman features of the T1 site.

3.5. SERS (Surface-Enhanced Raman Scattering) Spectroscopy

Raman spectroscopy has many advantages, such as rapid responses and the need for a very small sample volume, but the low intensity of Raman scattering is clearly a limit for many practical applications, and, therefore, finding means of enhancing the Raman process is often beneficial. In the 1970s, it was discovered that when molecules are adsorbed onto or near to corrugated metal surfaces, such as silver or gold nanoparticles [88], the intensity of the Raman signal coming from the molecules is greatly enhanced (by factors up to 10⁸ or even larger). It is now well recognized that this enhancement is mainly due to surface plasmons generated in the metal resulting in an evanescent field that interacts with and amplifies the Raman modes (electro-magnetic enhancement) [89,90,91]. Since its discovery, SERS has been employed in a plethora of cases, also enabling single-molecule (SM) SERS, and its use is continuously boosted by the ability to realize very specific corrugated structures. SERS has also become very popular in laccase-related studies [88,89,90,91,92].

In a study conducted by Michota-Kaminska et al. [93], vibrational spectroscopies, particularly SERS, were studied to evaluate the immobilization of laccase on gold and silver surfaces coated with self-assembled monolayers of thiols. This immobilization was verified by Raman spectra of the colored oxidation product of syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehydeazine). In Figure 10, some of the results obtained by Michota-Kaminska et al. [93] are reported. In particular, on the left, there is the absorption spectrum of the oxidized form of syringaldazine, with a maximum signal at about 530 nm. On the right, the SERS spectra are presented when laccase is absorbed on the Ag surface (a) and after the addition of syringaldazine on the Ag/laccase surface (b). The Raman and SERS spectra reported in Ref. [93] were obtained with 514.5 nm laser excitation. The results reported on the right of Figure 10 confirm the advantages offered by SERS. In this figure, SERS spectra confirmed the immobilization of laccase on silver surfaces coated with self-assembled monolayers of thiols differently from Raman spectra, which did not give evidence of the laccase immobilization.

Further examples of the use of Raman and SERS spectroscopy for the study of immobilized laccase are cited in the Supplementary Materials.

4. Physico-Chemical Properties of Phenolic Compounds

Phenolic compounds are part of the so-called secondary metabolites [94] and possess numerous abilities, including antimicrobial, antioxidant, and anti-inflammatory abilities. They consist of a phenolic moiety; therefore, the phenol itself is an organic compound that contains a hydroxyl (—OH) group attached to a carbon atom in a benzene ring (Figure 11) [95].

Phenolic compounds are synthesized in plants and play a key role in defense against external attacks [96]. Depending on the phenolic units present, they can be distinguished into simple phenols or polyphenols. These include coumarins, lignins, tannins, phenolic acids, and flavonoids [97]. There is much information in the literature on the bioactivity of phenolic compounds in vitro, but there is a lack of information on their action in vivo [94]. Interest in these compounds covers many areas from pharmacology [98] to textiles [99]. The favorable role of phenols is linked to the consumption of fruit, vegetables, and vegetable beverages in the human diet, but how the increasingly progressive and rapid production of these phenols is causing an increase in hazardous waste has also been observed [94]. The release of some phenols into wastewater and the resulting contamination of drinking water has caused attention to be focused on this problem. In this regard, both the European Commission (EC) and the U.S. Environmental Protection Agency (US EPA) (https://www3.epa.gov/region1/npdes/permits/generic/priority/pollutants.pdf (accessed on on 22 September 2023) have classified these compounds as priority pollutants, and special attention has been paid to phenolic compounds such as bisphenol A, catechol, resorcinol, and hydroquinone, whose structure is reported in Figure 12. These phenolic pollutants can cause muscle weakness, damage to the central nervous system, and the promotion of the formation of tumors. In Table S1, the permissible concentration limits of phenolic compounds are reported for food and environmental applications.

BPA is a synthetic compound with endocrine activity and is one of the most heavily produced substances in the world, being involved, for instance, in the production of polycarbonate plastics and epoxy resins [100]. BPA is used in the production of milk containers for children and water bottles. Exposure to BPA occurs primarily through food, due to the consumption of food and beverages contained in recycled bottles, epoxy-coated cans, and polycarbonate containers in which BPA has leaked [101,102,103,104]. Biomonitoring studies around the world have shown that more than half of the examined cases have exposure to BPA [104].

Resorcinol can be obtained by distillation of Brazilian wood extract or by synthesis and fusion processes [105]. The production of resorcinol mainly takes place in the rubber industry. The most evident exposure is at the cosmetic level, via hair dyes and dermatological products.

Catechol (or pyrocatechol, 1,2-dihydroxybenzene) is a crystalline compound, with two hydroxyl groups binding to a benzene ring in the orto-position [106]. It was first obtained in 1839 by Reinsch through the dry distillation of catechin, and it is now produced industrially from phenol. Although there is information on this compound in the literature, there is still no clear idea of its toxicity [107].

Hydroquinone is the main metabolite of benzene, with two hydroxyl groups binding to a benzene ring in the para position [106]. It is obtained by oxidation processes or by alkylation of benzene with propylene to isolate the para isomer [108,109]. An oxidation process with oxygen then takes place and the reaction product is treated with an acid to produce hydroquinone and acetone [109]. This phenol is used in various fields, including as a photographic developer or in motor fuels. Hydroquinone, resorcinol, and catechol, which are the result of the oxidation process of phenol, are more dangerous and less degradable than the latter as they increase the toxicity of the water [110]. It is therefore essential to monitor the levels of these compounds in samples derived from wastewaters to develop strategies for their removal [111,112,113]. For monitoring, the most used techniques are chromatographic ones, but they require too high costs and large amounts of time, and they do not allow in situ measurements [114]. Research is focusing on developing more accurate, sensitive, and less expensive sensing methods.

5. Laccase-Based Sensor Development

The above-described peculiar optical properties of laccase offer remarkable possibilities for developing different biotechnological applications as assays for evaluating laccase activity. Laccases and phenol reaction products show relevant optical signals in the UV and visible range that are usually employed for developing methods for monitoring laccase activity using absorbance or fluorescence measurements [115,116]. The optical properties of laccase are also exploited for elaborating methods for monitoring phenol degradation [117]. However, the most relevant use of optical characteristics of laccases is related to the design of optical biosensors that present significant advantages since they are small, light, chemically inert, non-toxic, and immune to electromagnetic interferences. The use of laccase allows the development of sensors for medical applications, essentially related to the detection of dopamine, catecholamines and other neuromodulator molecules [118,119,120,121,122]. Many optical laccase-based biosensors have been developed to detect and quantify phenolic compounds. Due to the dual role of phenol compounds [12], laccase optical biosensors have essentially been developed for monitoring phenol concentrations for environmental or dietary applications.

In the biotechnological use of enzymes, a fundamental role is played by immobilization procedures that have been briefly summarized in Supplementary Materials. An optical biosensor presents an active element consisting of laccase suitably immobilized in a matrix interacting with a solution containing the analyte of interest and generating an optical signal proportional to the analyte concentration. In the case of optical biosensors, the matrix should be optically transparent, able to preserve the catalytic activity of the immobilized enzyme and grant the diffusion of the analyte of interest.

In 2015, M.M. Rodriguez-Delgado et al. [13] revised the laccase-based biosensors for the detection of phenolic compounds and described the different optical biosensors reported in the literature. In this section, we intend to update their report by describing other developed laccase-based sensors by considering the different optical sensing schemes.

(a)

Sensor based on optical absorption spectroscopy.

The most used approach in developing biosensors using laccase is certainly the one related to the measurement of changes in absorption characteristics of the active element. The first optical biosensor that exploited the changes in absorption due to the interaction between immobilized laccase and phenol compound was proposed by Simkus and coworkers in 1996 [123]. The prepared fiber optic biosensor used a sol–gel matrix for immobilizing the enzyme and investigated the changes occurring in the absorption spectra due to the oxidation of 2,5-dimethoxy phenol in the presence of laccase. The intensity changes at 470 nm have been used to quantify the presence of the above-mentioned phenol in a concentration range equal to 1–60 mM. The authors also monitored the response time and time stability of their biosensors. It is worth noting that sol–gel technology offers the possibility to prepare optically transparent matrices useful for immobilizing enzymes. This technology offers the possibility of fabricating matrices for laccase immobilization with suitable chemical stability, optical transparency, and porosity (see Refs. [124,125,126], and the Supplementary Materials of the present paper for further information).

Abdullah et al. [127] designed an optical biosensor taking advantage of the ability of laccase to oxidize methoxy phenols in the presence of 3-methyl-2 benzothiazolinonehydrazone (MBTH) to produce azo-dye compounds for detecting phenols in environmental monitoring. Stacked films of MBTH in hybrid Nafion/sol–gel silicate and laccase in chitosan have been adopted for the preparation of a biosensor that is particularly sensitive to catechol, in comparison with other examined analytes, such as guaiacol, o-cresol, and m-cresol, that have also been considered by the authors. In Figure 13, the response curve of the biosensor is reported for increasing concentrations of catechol in phosphate buffer solution at pH 6.0. As is evident, the linearity range is equal to a 0.5–8.0 mM catechol concentration. A detection limit equal to 0.33 mM is evaluated. The detection limit has been estimated according to Refs. [128,129].

The same approach used by Simkus et al. [123] has been adopted for preparing a biosensor to determine the concentration of hydroquinone, resorcinol, and catechol. Laccases and phenol reaction products show useful optical signals in the UV and visible range. These signals have been adopted for biosensing applications since the compounds produced by the laccase interaction with different phenols are characterized by different optical absorption spectra. The occurrence of these different spectra increases the specificity of laccase-based biosensors [42], and they have been used to determine hydroquinone, resorcinol, and catechol concentrations, respectively (see Figure 14). In Figure 14, the absorption spectra of laccase solutions in the presence of the three phenol above-mentioned compounds are presented: (a) for hydroquinone, (b) for resorcinol and (c) catechol. For developing useful biosensors, laccase was immobilized in sol–gel matrices as described in Refs. [130,131]. On the right side of Figure 14, the calibration curves of sol–gel immobilized laccase at increasing concentrations of resorcinol and catechol are shown.

The hydroquinone–laccase reaction is characterized by a slow response time that prevented the use of this sensing scheme that was successfully used for resorcinol and catechol. Linear ranges up to 1.4 and 0.2 mM and an LOD of 4.5 and 0.6 µM have been obtained for resorcinol and catechol, respectively. Larger linear ranges, significant sensitivities, and good LODs are obtained with this biodevice in comparison with other biosensors using laccase from Trametes versicolor, as can be seen from Table 4 in Ref. [131]. This biosensor was also used with tap water samples added with known amounts of catechol and resorcinol for testing biosensing performances with real samples.

Sanz et al. [132] developed a laccase-polyacrylamide sensor exploiting the absorption and fluorescence properties of the enzyme and characterized by a linear range of 0.11–2.5 mM and a limit of detection (LOD) of 100 µM for phenols. This biosensor was also challenged with wastewater samples.

Table 1. Working characteristics for laccase solution and laccase-polyacrylamide sensor when ΔAbs and V₀ parameters are employed. Data are given in Ref. [132].

	Linear Range (M)	Slope	% RSD	LOD (M)	LOQ (M)
ΔAbs a	9.79 × 10−6 −7.50 × 10−4	4.20 × 103	1.07	2.94 × 10−6	9.79 × 10−6
V0 a	1.36 × 10−4 −5.00 × 10−3	1.70 × 101	2.61	4.07 × 10−5	1.36 × 10−4
ΔAbs b	1.09 × 10−4 −2.50 × 10−3	5.40 × 101	3.11	3.27 × 10−5	1.09 × 10−4

^a In solution with (Lac)_solution = 1.56 × 10⁻⁵ M; ^b in the sensor film with (Lac)_{sensor film} = 87.2 IU.

reports the results obtained for an enzyme solution (equal to 1.56 × 10⁻⁵ M) and the laccase immobilized in a polyacrylamide film working at pH 6 when the absorption properties of laccase are exploited. In Table 1, the performances of the working parameters are defined as

$$\begin{array}{c}\mathsf{\Delta }Abs=Ab{s}_{\lambda max,t}-Ab{s}_{\lambda max,0}\end{array}$$

(1)

$$V_0=\frac{\mathsf{\Delta }Ab{s}_{\lambda max}}{\mathsf{\Delta }t}=\frac{Ab{s}_{\lambda max,t}-Ab{s}_{\lambda ,0}}{t}$$

(2)

where Abs_λmax,0 and Abs_λmax,t represent the absorbance at a time t or 0 at the working wavelength.

Jedrychowska et al. [133] used laccase from Cerrena unicolor immobilized in low-temperature co-fired ceramics (LTCC) by physical adsorption to develop a valuable optical biosensor for monitoring of phenolic species in water in a continuous way. LTCC technology is a consolidated technique that is generally employed in the industry.

LTCC materials show very good physical and chemical properties, and three-dimensional structures can be readily fabricated. In a single LTCC multilayer substrate, optoelectronic components and systems for testing micro- and nanoliter volumes of samples can be incorporated [134,135]. The various steps necessary for fabricating the biosensor and for optimizing the different working parameters are described by Jędrichowska et al. [133]. Scanning electron microscopy has been employed to investigate the morphology of the laccase layer deposited on chemically modified supports. Sensor performances have been investigated in a flow-through system by exploiting the optical absorbance changes that occur when various concentrations of standard laccase assay substrate 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)-ABTS have been used. The contribution of various sensor working parameters (flow rate, optical source characteristics, and reproducibility) was studied. In their paper, Jedrychowska et al. [133] demonstrated that optical sensors with excellent working parameters (sensitivity, precision, and linearity) and simplicity of construction can be fabricated by using LTCC technology.

In Figure 15, the linear range obtained by considering the absorbance changes versus different concentrations of ABTS is shown. The R² coefficient for the linear range is close to 1 (R² = 0.975), indicating good accuracy of the fabricated sensor in a concentration range varying from 57 to 171 µM.

An innovative optical biosensor for determining the catechol concentration has recently been presented by Cano-Raya et al. [136]. Laccase from Trametes versicolor is bonded to anionic polyamide 6 (PA6) porous microparticles integrated into a Pebax MH1657 polymer binder that includes MBTH that gives a colored product when it interacts with the o-benzoquinone produced by the enzymatic reaction of catechol. The catechol concentration was determined by evaluating the changes in absorbance at 500 nm. In Figure 16, the calibration plot obtained for the developed biosensor is reported, and an LOD equal to 11 µM and a linear range up to 118 µM have been obtained.

The sensor was also tested using water samples from rivers and springs spiked with different concentrations of catechol, and it shows a recovery rate varying in the 97–108% interval.

Sorrentino et al. [137] developed an innovative optical biosensor using a laccase-based chimeric protein. Laccase from Pleurotus ostreatus was combined with hydrophobin layers to improve the immobilization efficiency of laccase. The chimeric protein was immobilized into multi-well plates without any purification step and used for monitoring two phenolic compounds: L-DOPA, which is a relevant compound in the pharmaceutical field; and caffeic acid, which is present in food [137]. The calibration curves for these two compounds have been constructed using changes in the absorption at 420 nm using ABTS as a substrate and the analytes as competing inhibitors in the ABTS oxidation due to laccase in indirect assays. The working parameters of these optical biosensors are reported in

Table 2. Working parameters of the optical biosensors developed by Sorrentino et al. in Ref. [137].

Analyte.	Sample	Linearity Range (µM)	LOD (µM)
L-DOPA	Buffer	5–1000	3
	Plasma	10–1000	2
Caffeic acid		50–4000	1

.

The biosensor was also challenged with spiked real samples: L-DOPA in plasma samples and caffeic acid in different beverages, such as tea and fruit juices. In both cases, high efficiency in analyte quantification was obtained [137].

(b)

Sensors based on fluorescence spectroscopy

The first attempt to exploit the fluorescence properties of laccase has been ascribed to Papkovsky et al. [138] in 1993. They aimed to investigate the performance of a solid-state fiber-optic oxygen sensor and design a new approach to enzymatic sensing using flow-injection analysis. The authors developed a simple and robust laccase-based sensing system that is useful for practical applications. In Figure 17, the scheme of the Papkovsky’s enzyme sensor is reported. The sensor was used for determining the concentration of different phenolic substances (Hydroquinone, phenol, 4-chlorophenol, 2,3-dichlorophenol, and 2,3,4-trichlorophenol) and real commercially available samples (Lipton “Yellow Label” tea (UK), Duncans “Double Diamond Leaf Tea” (India), Yun Nan black tea (China), “C.T.C”. granulated tea (India), and Caykur Rize tea (Turkey)). The Papkovsky et al. paper [138] reported the calibration curve for catechol and the quantitation of the polyphenol content in the different above-mentioned teas. The working characteristics of the fiber-optic flow-cell laccase sensor are summarized in Table 1 of Ref. [138] and reported in

Table 3. Relevant working parameters for optical sensors for the determination of phenolic compounds in environmental applications are discussed in the present paper (E and D stand for the use related to environmental, dietary, applications, respectively).

Phenolic Compound	Optical Technique	Applications	Sensitivity	Linear Range	LOD	Response Time	Ref.
Phenols	Absorption spectroscopy	E			0.1 M	Several hours	[123]
Catechol	Absorption spectroscopy	E		0.5–8.0 mM	0.33 mM	10 min.	[127]
	Time-course absorption spectroscopy	E	0.521 ± 0.016 Min−1 mM−1	Up to 0.2 mM	0.6 μM	3 min	[131]
	Absorption spectroscopy	E		Up to 118 μM	11 μM	30 min	[136]
	Absorption spectroscopy	E		9.79–750 mM	0.109 mM		[132]
ABTS	Absorption	E		From 60 μM to 180 μM	6.25 μM, 8.0 μM and 10.0 μM		[133]
Resorcinol	Time-course absorption spectroscopy	E	0.075 ± 0.001 Min−1 mM−1	Up to 1.4 mM	4.5 μM	3 min	[131]
Caffeic acid	Time-course absorption spectroscopy	D, E		50–4000 μM	1 μM		[137]
Polyphenols	Fluorescence	D		0–7 mM	0.2 mM (or 2 nmol)	2–3 min	[138]
Polyphenols	Fluorescence	D		Catechol (0.08–5 µmolL−1) Hydroquinone (0.05–2 µmolL−1) Hydroxyhydroquinone (0.09–5 µmolL−1) Gallic acid (0.13–5 µmolL−1) Pyrogallol (0.17–5 µmolL−1)	Catechol (0.01 µmolL−1) Hydroquinone (0.01 µmolL−1) Hydroxyhydroquinone (0.03 µmolL−1) Gallic acid (0.04 µmolL−1) Pyrogallol (0.04 µmolL−1)		[139]
Polyphenols	Fluorescence	D			1 ng/mL		[140]
Polyphenols	Fluorescence	D	(8.9 + 0.2) 102 mM−1	Up to 200 µM	7.4 µM		[141]
Catechol	Phase angle	D, E		40–600 µM	0.04 mM	25 min	[142]

.

Andreu-Navarro et al. [139] developed a significant approach for the optical detection and quantification of different polyphenol compounds (catechol, gallic acid, hydroquinone, hydroxyhydroquinone, phloroglucinol, pyrogallol, and resorcinol) that can be present in beverages. The described biosensor takes advantage of the inhibition of the green indocyanine fluorescence induced by polyphenols in the presence of laccase and gold nanoparticles with positive charges. Laccase fluorescence rapidly decreases when the fluorophore is added. The decrease can be ascribed to the catalytic effect of the enzyme on fluorophore oxidation. The fluorescence decrease effect is slower proportionally to the concentration of the present polyphenols.

Figure 18 shows the experimental apparatus used, which includes a device for the rapid mixing of reagents. A solution of laccase and gold nanoparticles was placed in one of the syringes; in a second syringe, the sample was placed in a solution with indocyanine green. A third syringe was used for flow control. The fluorescence was excited at 764 nm and is collected at 806 nm using a spectrofluorometer, which allows us to follow the time course of the fluorescence on the time scale of seconds. The spectrofluorometer software used for the treatment of kinetic measurements was adopted for acquiring and processing data. To quantitatively determine the analyte, the authors used the difference in the necessary time to observe a decrease in the fluorescence signal in the presence and absence of the analyte, as shown in Figure 19. In this figure, the slope of the curve obtained in the presence and the absence of the polyphenol has practically the same value, indicating that the oxidation kinetics do not influence each other. The present biosensor allowed catechol to have an LOD of 0.01 mM and a linear range of 0.08–5 mM and has been used for the determination of the concentration of polyphenols in beverages such as tea and fruit juices.

The proposed system suffers from the limit of enzyme consumption being higher compared to the normal technological use of enzymes, which provides for their immobilization; however, the consumption is limited. Furthermore, they use positively charged gold nanoparticles to modulate the timing of enzyme kinetics, by exploiting the nanoparticle’s ability to reduce enzyme activity.

Akshath et al. [140] proposed an optical biosensing scheme with a different use of the laccase enzyme. They exploited the conversion mechanism from polyphenols in mono/polyquinones induced by laccase for quenching CdTe quantum dot fluorescence. The resulting mono/polyquinones can quench quantum dot fluorescence as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects and by a supposed charge transfer process from quinones to QDs. This process, illustrated in Figure 20, was used for detecting and quantifying polyphenols that can detect these analytes in the 1 ng/mL–100 mg/mL range with an LOD equal to 1 ng/mL. The authors observed that quenching of QDs fluorescence is linear with polyphenol concentration, so a differential quenching response provided sensitive “fingerprint” detection of individual polyphenols [140].

The developed sensor has been also applied to real samples obtained by a water solution of plant extract spiked with various polyphenols. The added quantities have been recovered in a very efficient way, as shown in Table 2 of Ref. [115]. The authors also demonstrated that there are no interference effects due to metal ions like aluminum, copper, zinc, iron, etc., which can be present in small quantities in plant extracts.

Recently, Mediavilla et al. [141] developed a fluorescence-based assay for evaluating the total polyphenol concentration in beverages using laccase from Trametes versicolor and carbon nanodots (CD). Using this bioconjugate, it was possible to monitor the enzymatic reaction thanks to the decrease in fluorescence signal due to the quinone quenching effect. This method has been successfully applied for the determination of the total concentration of polyphenols (represented as gallic acid concentration) in wine, fruit juice, and rice leaf extract spiked samples.

Another fiber-optic-based laccase biosensor for polyphenolic compound detection has been developed by Bilir et al. [142]. They used laccase from Pleorotus ostreatus that was immobilized on the surface of a commercially available fiber optic oxygen sensor. Tetramethyl orthosilicate (TMOS), trimethoxymethylsilane (Tri-MOS), and polyvinyl alcohol (PVA) were added to the immobilized laccase region as a diffusion layer. In Figure 21, the sensing mechanism is shown.

The detection mechanism exploits the luminescence emission from oxygen-sensitive dyes contained in sensing spots. After excitation, the interaction between the luminescent dyes and an oxygen molecule causes the transfer of the excess energy to that molecule, exploiting a dynamic quenching process (Figure 21). The light emission intensity from sensing spots and the average excitation lifetime decreased. The excitation lifetime and oxygen concentration are related by the Stern–Volmer relationship, and the phase modulation technique is applied for precise measurements [143]. The sensor spot is excited with sinusoidally modulated light, and the emitted light is also sinusoidally modulated. The average excitation lifetime is then detected as the phase angle between these two optical signals. The measurements are carried out in a flow-through system.

The Bilir et al. [142] biosensor is characterized by a large linear range for catechol (40–600 μM), as shown in Figure 22. The other working parameters are reported in Table 3. In agreement with the authors, the designed biosensor can be easily produced and is reproducible and stable. The authors propose its use in the food industry as well as in environmental monitoring for the detection of phenolic compounds.

Well-known optical biosensors are characterized by high sensitivity and offer significative advantages in terms of sensitivity, linear range and LOD. They are also chemically inert, effective for remote sensing, and immune to electromagnetic interference. Also, laccase-based biosensors profit from these advantages, but some of them can suffer from the low specificity that characterizes this enzyme [3,144,145]. However, some immobilization procedures or other strategies can be adopted to obtain an increase in the specificity towards phenolic substrate [8,146]. In order to compare the performances of the different biosensors described above, we reported their main working parameters in Table 3, which provides an overview of different approaches for the detection of phenolic compounds through the development of spectrophotometric and fluorescence sensors for environmental and food applications. This table reports information concerning the main biosensors’ working parameters, including the following: sensitivity, linearity range, limit of detection (LOD) and response time. The optical absorption-based approach constitutes the preferential choice of allowing an LOD of a few µM with a response time equal to a couple of minutes. Concerning florescence-based biosensors, they show comparable performances; that said, in recent years, the use of nanotechnologies has allowed the design and fabrication of highly performing optical biosensors [140,141].

The optical methods shown in Table 3 are among the most widely used, but more innovative techniques, such as those based on SERS, are emerging. In recent years, great interest has been shown in the development of nanozymes that mimic natural enzymes. The development of these nanomaterials permits overcoming the drawbacks that characterize natural enzymes such as a high price, poor stability, and difficulty to recycle. Nanozymes can have a low price, high stability, and tailored activity. In addition, they can be largely produced and stored for a long time [147,148]. Thus, nanomaterials with laccase activity can represent ideal candidates for developing innovative optical sensors [149].

6. Conclusions

The relevance of laccases in many different contexts related to textile and paper industries, wastewater treatment, pharmaceutical production, fuel cell fabrication, and biosensor development motivates the intense investigation of their structural, biochemical, and physical properties. In this review, we focused on the optical properties of laccases and their application to the designing and fabrication of optical biosensors for phenolic compounds that are ubiquitous in our lives. We revised the literature concerning UV–Vis absorption and fluorescence, FT-IR, Raman, and SERS spectra of different kinds of laccases and presented the different approaches used for exploiting the optical properties of laccases for detecting and quantifying phenolic compounds. We also reported a brief description of the main characteristics of these compounds and different kinds of laccases. Since the practical use of enzymes strictly requires their immobilization in suitable supports, a short description of the basic principles of these procedures is reported in the Supplementary Materials. By considering the different laccase optical biosensors reported in Table 3 and the advanced optical nanotechnologies that are nowadays available, it is evident that there is a large space for developing sensitive, fast, and simple biosensors and new physical and chemical sensing schemes for phenolic species detection and quantification.