Phenol biosensor based on electrochemically controlled integration of tyrosinase in a redox polymer

An amperometric biosensor for the detection of phenolic compounds was developed based on the immobilization of tyrosinase within an Os-complex functionalized electrodeposition polymer. Integration of tyrosinase within the redox polymer assures efficient catechol recycling between the enzyme and the polymer bound redox sites. The non-manual immobilization procedure improves the reproducibility of fabrication process, greatly reduces the desorption of the enzyme from the immobilization layer, and, most importantly prevents fast inactivation of the enzyme by its substrate due to fast redox cycling. A two-layer sensor architecture was developed involving ascorbic acid oxidase entrapped within an electrodeposition polymer in a second layer on top of the redox polymer/tyrosinase layer. Using this sensor architecture it was possible to eliminate the current interference arising from direct ascorbate oxidation up to a concentration of 630 μM ascorbic acid. The effects of the polymer thickness, the enzyme/polymer ratio, and the applied potential were evaluated with respect to optimal sensor properties. The sensitivity of the optimized sensors for catechol was 6.1 nA μM⁻¹ with a detection limit of 10 nM, and for phenol 0.15 nA μM⁻¹ with a detection limit of 100 nM.     

Development and optimization of a solid composite tyrosinase biosensor for phenol detection in flow injection systems

Bulk-modified epoxy-graphite tyrosinase biosensors were fabricated by four different procedures. The influence of these fabrication procedures on the analytical performance of the enzyme electrode in an amperometric wall-jet flow cell has been studied. The bioprobe performance is assessed by cyclic voltammetry. Higher current densities and narrower peaks were obtained when the enzyme was introduced in the dry state into the epoxy-graphite material, instead of introducing it previously dissolved in the buffer. In the F1 system responses of 11.79 μA cm⁻² and 1.43 μA cm⁻² are then obtained for catechol and phenol respectively for 50 μL injections of 20 μM solutions. Moreover, if gold/palladium is introduced into the epoxy-graphite, a further increase in current is achieved resulting in 27.70μA cm⁻² and 4.90μA cm⁻²for catechol and phenol, respectively. This biosensor can operate in aqueous as well as in mixed aqueous-organic environments.     

Optimisation and validation of an automated solid phase extraction technique coupled on-line to enzyme-based biosensor detection for the determination of phenolic compounds in surface water samples

Summary A fully integrated screening system for phenolic compounds was developed incorporating on-line solid phase extraction, fractionation and biosensor detection. Two different types of biosensors, solid graphite and carbon paste electrodes incorporating the enzyme tyrosinase, were compared and used in the screening system. Interfacing of the solid phase extraction and fractionation with the biosensor detection was given special attention since the biosensors were not compatible with the organic modifier used for desorption of phenols from the solid phase extraction step. The system was validated with conventional analytical techniques. Surface water samples from the Ebro river were spiked with 1,10, and 25g L^–1 of catechol, phenol,p-cresol, respectively. Three out of seven samples were spiked and the correct samples were identified, containing phenols equivalent to the spiked concentrations.     

Pure Organic Phase Phenol Biosensor Based on Tyrosinase Entrapped in a Vapor Deposited Titania Sol‐Gel Membrane

A novel amperometric biosensor was constructed for the determination of phenols in pure organic phase. This biosensor was fabricated by immobilizing tyrosinase in a titania sol‐gel membrane which was obtained with a vapor deposition method. This method was facile and avoided the calcination step needed in conventional titania sol‐gel process. The titania sol‐gel membrane could effectively retain the essential water layer around the enzyme molecule needed for maintaining its activity in organic phase. The experimental parameters such as solvent and operating potential were optimized. At ?100 mV this biosensor showed a good amperometric response to phenols in pure chloroform without any mediator and rehydration of the enzyme. For catechol determination the sensor exhibited a fast response of less than 5 seconds. The sensitivity of different phenols was as follows: catechol > phenol > p‐cresol. Additionally, the apparent Michaelis‐Menten constants of the encapsulated tyrosinase to catechol, phenol and p‐cresol were found to be 0.15±0.003, 0.17±0.008 and 0.21±0.004 mM, respectively. The biosensor had also good reproducibility and stability. This work provided a promising platform for the construction of pure organic phase biosensors and the determination of substrates with poor water solubility.     

Intercalation assembly of kojic acid into Zn-Ti layered double hydroxide with antibacterial and whitening performances

The inhibitor of melanin and the bacteriostatic agent kojic acid was inserted into Zn-Ti layered double hydroxide (LDH) by anion-exchange reaction. The structure, slow release, antibacterial and skin whitening activity were studied.     

A sensitive mediator-free tyrosinase biosensor based on an inorganic-organic hybrid titania sol-gel matrix

A novel inorganic-organic hybrid titania sol-gel nanocomposite film was prepared to fabricate a sensitive tyrosinase biosensor for the amperometric detection of trace phenolic compounds without additional electron mediators. Acetylacetone worked as a complexing ligand to chelate with Ti atom in the synthesis process, and the pH of the titania solution could be adjusted to the value which was optimum for retaining tyrosinase activity and such a membrane was stably attached on to the surface of a glassy carbon electrode (GCE). This titania matrix could supply a good environment for enzyme loading, which resulted in a high sensitivity of 15.78 μA μM⁻¹ cm⁻² for monitoring phenols with a detection limit of 1×10⁻⁸ M at a signal-to-noise ratio of 3. The TiO₂ sol-gel derived biosensor exhibited a fast response less than 10 s and a good stability for more than 2 months.     

Ionic liquid–modified cellulosic hydrogels for loading and sustained release of selenourea: an ensuing inhibition of tyrosinase activity

Cellulose-based hydrogel materials were prepared and modified with tannic acid and l-methionine using ionic liquid as the solvent. The gels were prepared to develop a sustained release medium for selenourea (SeU). The drug delivery characteristics of selenourea-loaded cellulose (CSeU), selenourea-loaded tannic acid-modified cellulose (CTSeU), and selenourea-loaded L-methionine-modified cellulose (CMSeU) were investigated in aqueous media and simulated gastric fluid (SGF) media. This modified gel beads have been characterized using field emission scanning electron microscope, X-ray energy-dispersive spectroscopy, Fourier transform infrared spectroscopy, thermogravimetry–differential thermal analysis and swelling properties and compared with those of the unmodified ones. We also investigated the inhibitory effects of SeU released from these gels on the activity of mushroom tyrosinase. Out of all the gel materials, CTSeU showed maximum SeU release both in water and SGF media. However, tyrosinase inhibitory action in PBS medium was comparable for all the three gel materials.     

Oxygen Dependence in a Dual‐Phase Electrochemical Biosensing System

Oxygen dependence of a tyrosinase‐based electrochemical biosensor for determination of phenol in aqueous and organic media was systematically investigated. The result demonstrated that the enzymatic coupling reaction rate of tyrosinase (deoxy form) and O₂ to regenerate tyrosinase (oxy form) is a kinetically fast reaction, and the significant change of O₂ concentration in aqueous solution did not affect the coupling reaction. The further increase of O₂ concentration did not increase the overall oxidation reaction rate of the substrate at low substrate concentration (e.g.,<10 μM phenol) when O₂ concentration was greater than 8.9 ppm. The oxygen dependence was observed in the case of high substrate concentration due to insufficient amount of O₂ available for the regeneration of tyrosinase. In other words, the upper linear range is oxygen dependent for tyrosinase biosensors. The phenol biosensors employing microelectrodes had wider upper linear ranges than macroelectrodes in both aqueous and organic phase, which can be explained by the oxygen dependence.     

Screen-printed multienzyme arrays for use in amperometric batch and flow systems

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.