Phenols, Tyrosinase & Laccase - Microbial biosensors

Caffeic acid is a kind of phenolic acid, which has been found to be pharmacologically active as an antioxidant, antimutagenic, anticarcigenic agent, lipoxygenase inhibitor and it has also antibacterial, antiinflamatory and styptic activities. Ferulic acid arises from the metabolism of phenylalanine and tyrosine. It is the most abundant hydroxycinnamic acid in the plant world and occurs primarily in seeds and leaves both in its free form and covalently linked to lignin and other biopolymers. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Due to its phenolic nucleus and an extended side chain conjugation, it readily forms a resonance stabilized phenoxy radical which accounts for its potent antioxidant potential. UV absorption by ferulic acid catalyzes stable phenoxy radical formation and thereby potentiates its ability to terminate free radical chain reactions.

By virtue of effectively scavenging deleterious radicals and suppressing radiation-induced oxidative reactions, ferulic acid may serve an important antioxidant function in preserving physiological integrity of cells exposed to both air and impinging UV radiation. Similar photoprotection is afforded to skin by ferulic acid dissolved in cosmetic lotions. Its addition to foods inhibits lipid peroxidation and subsequent oxidative spoilage. By the same mechanism ferulic acid may protect against various inflammatory diseases.

A number of other industrial applications are based on the antioxidant potential of ferulic acid. Nowadays, the determination of phenolic compounds is of great importance, since they are widely used in industrial processes, such as the manufacture of plastics, polymers, drugs and dyes.

These kinds of compounds are also result as break-down from some pesticides and by-products from paper pulp industry, with the types and abundances of phenolic compounds changing with the particular source or mill process. Phenolic compounds belong to a class of polluting chemicals that are easily absorbed by animals and humans through the skin and mucous membranes. Their toxicity affect a great variety of organs and tissues, primarily lungs, liver, kidneys and genito-urinary system.

Certain phenols and related aromatic compounds are highly toxic, carcinogenic and allergenic and due to their toxic effects, their determination and removal in the environment are of great importance. In addition, due to their great variety, phenolic compounds show a broad range of toxicity levels, being phenol and its chlorinated or alkylated derivatives, classified as priority pollutants. Thus, the development of procedures for detection and simultaneous determination of these compounds in different matrices is highly desired.

Analytical methods for the detection and quantification of mixtures of phenols are usually based on analytical separation techniques, which allow the identification and quantification of individual constituents. Many methods have been developed for the determination of phenolic compounds, such as chromatographic, fluorimetric and spectrophotometric techniques. However, these techniques do not easily allow continuous monitoring, they are expensive, time-consuming, need skilled operators, and sometimes require preconcentration and extraction steps that increase the risk of the sample loss or contamination. Thus, the development of new methods that allows the simultaneous determination, without previous separation of these compounds is a relevant subject of research. Biosensors tend to be more stable and more suitable to environmental monitoring, clinical testing, or food assays compared with the direct electrochemical oxidation of phenols.

Recently, phenolic acid detection has been obtained in blood, wine, tea, fruit juice, oil and plant extracts by using HPLC with mass spectrometry, UV or electrochemical detection, capillary electrophoresis with UV or electrochemical detection, liquid chromatography, gas chromatography-mass spectrometry, fluorescence, electrochemical detection and biosensor. Compared with other methods, biosensors have the potential to overcome most of the disadvantages of conventional methods.

Amperometric biosensors have been prepared as a mediated and unmediated systems. A lot of types electrode combined with laccase have been developed for detection of phenolics. Very recently, it has reported that laccases are widespread in bacteria. Biosensors have found promising applications in various fields such as biotechnology, food and agriculture product processing, health care, medicine and pollution monitoring The combination of oxidoreductases and amperometric electrodes is by far the most commonly studied biosensor concept and through various strategies the enzyme reaction can be easily followed and sensitively measured by electrochemical means.

The success of an electrochemical sensing process relies mainly on a proper choice of the working electrode. Solid electrodes like gold, platinum and carbon based electrodes were utilized as electrode transducers. Apart from these, mercury thin film electrode (MTFE) based biosensor was developed and used for H2O2 detection. Mercury film electrode (MTFE) consists of a very thin film (10-100 μm) layer of mercury that is distributed over the support material. Glassy carbon electrode (GCE) is claimed to be most favorable support material because mercury plated glassy carbon is not contaminated by amalgamation and it does not diffuse into the support material. The mercury film formed on a GCE surface is composed of many droplets that lead to a lower hydrogen over voltage. Besides MTFE’s provide larger surface to volume ratio and can be utilized in different cell configurations Furthermore, as small quantities of mercury is used for the preparation of MTFE, the consumption of metallic mercury is minimized. SPGE (screen printed graphite electrode) has also been utilized as the support material for MTFE. These electrodes are based on screen-printing technology and can be employed as low-cost disposable electrochemical sensors. A laccase biosensor for the detection of phenolic compounds was obtained with both GCE and SPGE used as the support material for MTFE formation. Instead of oxygen, mediator can be used as an electroactive compound. Ferrocene and its derivatives that can serve as electron shuttling between enzyme active site and electrode have been used as redox mediators for the development of biosensors.

Phenolic acids are oxidized by laccase. When ferrocene is present in the reaction medium, it acts as an electron donor during oxidation of phenolic acids. Mediator is oxidized by the working electrode. As a result, a current is decreased and detected using the electrode system.

Conducting polymers have enough scope for the development of various sensors. Sensor systems based on conducting polymers also rely on sensible changes in the optical and electrical futures of this kind of materials.

Laccases (benzenediol: oxygen oxidoreductase, E.C. 1.10.3.2) are copper containing oxidoreductases produced by higher plants and microorganisms, mainly fungi. Laccases reduce oxygen directly to water in a four-electron transfer step without intermediate formation of soluble hydrogen peroxide in expense of one-electron oxidation of a variety of substrates, e.g. phenolic compounds Laccases have wide substrate specificity and a great potential for the determination of phenolic compounds and also show broad specificity in the process of oxidizing many compounds (mainly of phenolic type) and can be used for detoxification of a number of aquatic and terrestrial xenobiotics, industrial waste-waters, as well as for biotechnological treatment of industrial products. In the presence of mediators, laccase can also play a role in the oxidation of non-phenolic substrates. Rather low activity and stability of plant laccases, however, limited the application of laccases based biosensors. To become practical biosensors application a reality an inexpensive source of laccases must be obtained. Different sources of laccases were used to develop thick film sensors for the determination of phenolic compounds. Moreover, characterization and sample application of these sensors were also carried out.

The use of microbial biosensors to determine the concentrations of substances is based on the presence of specific enzyme systems in microorganisms which transform certain chemical compounds. The transformation processes can be accompanied by the appearance of electrochemically active products or utilization of reaction co-substrates, which enable the use of standard electrochemical techniques amperometry or potentiometry for their registration.

As judged by their sensitivity, time of response and stability of signals, microbial sensors are similar to enzyme based sensors but are less selective. This may be due to the complexity of the elements of the enzyme apparatus of cells. Insignificant amount of biomass as well as high stability make the use of microbial sensors preferable in some cases compared to enzyme sensors. This is especially true in the detection of a pool of toxic compounds showing similar composition, or the assessment of comprehensive indices of the condition of the environment as, for instance, biological oxygen demand (BOD),

Amperometric biosensors using bacterial cells were developed for the detection of phenolic compounds. Pseudomonas putida DSM 50026 was used as a biological component and the measurement was based on the respiratory activity of the cells. For this purpose, the cells were grown in the presence of phenol as the sole source of organic carbon. As well as phenol adapted cells, the bacterium which used the glucose as the major carbon source, was also used to obtain another type of biosensor for the comparison of the responses and specificities towards to different xenobiotics.

Polyphenols in wine and must

Polyphenols are secondary metabolites of plants and they are mainly present in berry skins and seeds, but almost absent in grapes’ juice. They are responsible for the sensory characteristics of wine such as colour, flavour, astringency and hardness. Both polyphenolic content and composition of wines depend on geographical, atmospherical, and a variety of factors regarding production systems. In red wines the content of polyphenolic compounds varies from 2 to 5 g/L on average, while in white wines it is about 100 mg/L. These compounds are essential nutritional elements and great relevance is given to their antioxidant properties, which have a definitely positive effect on human health; as a consequence, it is very useful to characterize a wine by its polyphenolic content. In a previous article was described the development of an amperometric biosensor for the determination of phenols in wines, based on screen printed graphite electrodes (SPEs), modified by mixing into the graphite’s ink a redox mediator, namely ferrocene. Tyrosinase enzyme was immobilized on the working electrode using different immobilization techniques. Laccase (p-diphenol oxidase containing copper ions) has been co-immobilized with Tyrosinase in a sol gel matrix of diglycerysilane (DGS) with the aim of widening the range of phenolic compounds detection due to the different catalytic activity of such enzymes. After a preliminary study of the bi-enzyme biosensor parameters and optimization of the suitable analytical conditions for measurements in Flow Injection Analysis (FIA), wines and samples of must, have been analyzed during the fermentation process. Spectrophotometric analyses of the samples, the Folin-Ciocalteu method and the measure of the absorbance of wines at 280 nm, have been carried out too in order to compare the results obtained with the biosensor with those ones obtained with reference methods. The interfering effect of sulphur dioxide, usually added during wine-making, in the determination of phenols by the presented biosensor is also briefly discussed.

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