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The catalyst ZeoSonFe(M1), mixed with hydrogen peroxide and UV light, presented the highest degradation percent within 120 minutes of treatment respect to ZeoSonFe(M2), ZeoSonFe(M3) and ZeoSonFe(M4) which accounted for 72.92, 84.27, and 72.30 %, respectively. The spectrum of UV-Vis degradation showed that the absorption main bands of IC (612 and 286 nm) decrease due to the rupture of C=C bond that belong to the chromophorous group IC. The catalyst ZeoSonFe(M1) was characterized with infrared (FTIR), Scanning Electron Microscopy (SEM) and pore diameter analysis with the BET method. To describe the kinetics of the oxidative reaction the pseudo first order model was applied.

Amperometric biosensors based on Laccase from Trametes versicolor (LTV) orTyrosinase from mushrooms(TM) weredeveloped and optimized for monitoring the phenolic compounds content in tea infusionsand grapes extracts. With the aim of comparing the performances of the biosensors developed using two different methods, TM was immobilizedfirstly bycross-linking with glutaraldehyde and afterward by entrapment in a polymeric film using polyvinyl alcohol photopolymer PVA-AWP (Azide-unit pendant water-soluble) both of them onto disposable graphite screen-printed electrodes (SPE), evaluating at the same time, different amount of enzyme fixed on the working electrode surface. Sincethe obtained results show thatthe performance of the TM-SPE manufactured by entrapment withinPVA-AWPis better than cross-linking, a different enzyme is evaluated also entrapped in the same polymeric film.LTVwasimmobilized onto disposable graphite screen-printed electrodes (SPE)to construct a second type of biosensor. Sensitivity optimization in terms of pH, temperature and applied potential was carried out. The linear range, detection limit, operational and storage stabilities were also determined.The laccase biosensor(LTV-SPE)was calibrated for o-, m-and p-diphenol as well as caffeic acid.The highest response was found at 0.1 M acetate buffer pH 4.7, though it must be added the good reproducibility and operational stability were also obtained. The useful lifetime of the biosensor is estimated to be greater than 6 months.Whereasthe tyrosinasebiosensor (TM-SPE)was calibrated only for its natural substrate o-diphenol (catechol) and caffeic acid at 0.1 M phosphate buffer pH 7.0.LTV-SPE was used for the determination of the equivalent phenol content in tea infusions whereas TM-SPE was used to determine the equivalent phenol content in grapes extracts, both of them by the direct additionof small sample into the electrochemical cell;the results were compared with those from the Folin-Ciocalteu spectrophotometric method.The amperometric detection exhibits some interesting advantages such as high simplicity, minimal sample preparation and shorter response time. A stable and sensitive amperometric response was obtained toward standard diphenolic compounds, grapes extracts and herbal infusions. In conclusion both types of biosensors are useful for easy and fast monitoring of equivalent phenol content that can be related to the antioxidant capacity of natural extracs. En este trabajo se describe el desarrollo y optimización de biosensores amperométricos basados en La casa de Trametes versicolor (LTV) o Tirosinasa de hongos (TM) ;así como su aplicación para determinar el contenido de compuestos fenólicos en tés y extractos de uvas. Los estudios electroquímicos realizados incluyen diferentes técnicas como voltamperometría cíclica (VC), voltamperometría de pulso diferencial (VPD) y amperometría, empleando SPE´s modificados con LTV o TM, ambas enzimas del grupo de las polifenoloxidasas (PPO). Con el propósito de comparar el desempeño de biosensores construidos usando dos métodos diferentes, TM fue inmovilizada primero por reticulado con glutaraldehído y posteriormente por atrapamiento usando alcohol polivinilico PVA-AWP (Azide-unit pendant water-soluble). En ambos métodos se utilizaron electrodos impresos de grafito (SPE)y se evaluaron diferentes cantidades de enzima sobre la superficie del electrodo de trabajo. Adicionalmente se analizaron y optimizaron otras condiciones experimentales en base al esquema del diseño factorial para 3 factores (temperatura, pH y potencial aplicado) y una vez determinadas las condiciones experimentales óptimas se realizó el análisis de extractos de uvas y distintas infusiones herbales. Los resultados muestran que el biosensor modificado con tirosinasa (TM-SPE) inmovilizada en PVA-AWP, supera al biosensor construido porreticulado. La enzima LTV también es estudiada utilizando los mismos métodos de inmovilización. Se realizó también la optimización de los biosensores en términos de pH, temperatura y potencial aplicado. Se determinó el intervalo de linealidad, límite de detección, estabilidad operacional y estabilidad al almacenamiento. El biosensor modificado con lacasa (LTV-SPE) fue calibrado usando o-, m-, p-difenol y ácido caféico. La respuesta más alta se encontró utilizando amortiguador de acetatos 0.1 M de pH 4.7y 30°C. El tiempo de vida del biosensor se estima en más de seis meses. Por otra parte el biosensor modificado con tirosinasafue calibrado únicamente para catecol y para ácido caféico, ambos en amortiguador 0.1 M de fosfatos pH 7.0. LTV-SPE fue utilizado para la determinación del contenido fenólico equivalente (CFE) en infusiones de tés mientras que el biosensor TM-SPE fue utilizado para determinar el CFE en extractos de uvas. El biosensor modificado con LTV muestra mayor sensibilidad y estabilidad en comparación con otros sensores enzimáticos reportados en la literatura. La principal ventaja de los dispositivos propuestos, radica en la facilidad del método de fabricación y de análisis pues las mediciones se realizan mediante la adición directa a la celda electroquímica de pequeños volúmenes de las muestras reales. En conclusión se considera que la inmovilización de las enzimas LTV y TM sobre SPE’s permite la construcción de biosensores que muestran buen desempeño analítico quepueden usarse como un método rápido y confiable para determinarel contenido fenólico, el cual puede vincularse a la capacidad antioxidante de los extractos naturales.

The working electrode of screen-printed-type electrodes, SPE, was modified with Tyrosinase enzyme (Tyr) using different immobilization methods namely: entrapment, using water soluble polyvynil alcohol, PVA, cross-linking using glutaraldehyde, GA, and cross-linking using GA and Human Serum Albumin, HAS. The resulting electrodes were named: SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/ HSA/GA, respectively. All these biosensors were characterized through amperometry, immersed in an aqueous solution containing different catechol concentrations, and EIS techniques. From the amperometric evaluation, the apparent Michaelis-Menten constant, Km´, of each biosensor were evaluated and from the impedance measurements the respective charge transfer resistance, Rct, were assessed. It was found that the biosensor SPE/Tyr/GA has the lowest Km´ (57 ± 7) μM and Rct values. Moreover, this electrode also displays both the lowest detection and quantification limits for catechol quantification, notwithstanding, the highest sensitivity corresponds to the SPE/Tyr/PVA biosensor. Using the SPE/Tyr/GA biosensor, the Trolox Equivalent Antioxidant Capacity (TEAC) was determined for infusions prepared with Mirto (Salvia microphylla), Hierba dulce (Lippia dulcis) and Salve Real (Lippia alba); medicinal plants commonly used in Mexico.

An amperometric biosensor based on laccase, from Trametes versicolor (LTV), was developed and optimized for monitoring the phenolic compounds content in tea infusions. The fungal enzyme was immobilized by entrapment within polyvinyl alcohol photopolymer PVA-AWP (azide-unit pendant water-soluble photopolymer) onto disposable graphite screen-printed electrodes (SPE). Sensitivity optimization in terms of pH, temperature and applied potential was carried out. The linear range, detection limit, operational and storage stabilities were also determined. The laccase biosensor (LTV-SPE) was calibrated for o-, m- and p-diphenol as well as caffeic acid. The highest response was found at 0.1M acetate buffer pH 4.7, though it must be added the good reproducibility and operational stability were also obtained. The useful lifetime of the biosensor is estimated to be greater than 6 months. LTV-SPE was used for the determination of the equivalent phenol content (EPC) in tea infusions by the direct addition into the electrochemical cell: the results were compared with those from the Folin-Ciocalteu spectrophotometric method. The amperometric detection exhibits some interesting advantages such as high simplicity, minimal sample preparation and shorter response time. A stable and sensitive amperometric response was obtained toward standard diphenolic compounds and herbal infusions. These biosensors are useful for easy and fast monitoring of EPC that can be related to the antioxidant capacity of natural extracts.

The immobilization of Laccase from Trametes versicolor (TvL) by means of entrapment, cross-linking and covalent bonding methods is analyzed through the development and characterization of amperometric screen-printed sensors for phenolic compounds. In order to do the immobilization with each of the methods stated, polyvinyl alcohol (PVA), glutaraldehyde (GA) and N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide clorhydrate (EDC), were used respectively; the electrochemical characterization was carried out at (30.0 ± 0.5) °C and pH = 4.70 ± 0.01. The apparent Michaelis-Menten constant, Km ´, for the enzymatic oxidation of hydroquinone, HQ, to p-quinone, Q, was used as comparison parameter, to provide information on the enzymatic kinetic parameters, as well as the analytic parameter linked directly to the biosensor. After comparing the different immobilization methods, it became apparent that the best one was cross-linking at 40 °C, that gave the lowest Km ´ ((170.1 ± 3.2) µM), and HQ limit of detection ((17.0 ± 6.6) µM) values. The antioxidant capacity of real samples, infusion of medicinal plants, was evaluated using this biosensor.