Covalent Immobilization Of Tyrosinase Research Articles

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening.

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibratingsamplemagnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13±0.91μM compared to kojic acid (41.96±0.78μM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.

Amperometric phenol biosensor based on covalent immobilization of tyrosinase on Au nanoparticle modified screen printed carbon electrodes

A highly selective and sensitive amperometric biosensor for the detection of phenol was developed based on a platform where Au nanoparticles (AuNPs) are electrodeposited onto a disposable screen printed carbon electrode and tyrosinase is then covalently immobilized on the AuNP's using alkanethiol and cross-linker molecules. The electrocatalytic responses of the tyrosinase modified biosensor for the detection of phenol were measured using both cyclic voltammetry and square wave voltammetry. Temperature, buffer pH and the amount of tyrosinase immobilized on the electrode surface were also optimized for phenol sensing. A high sensitivity of 15.7 µA ppm(-1), a low detectable phenol concentration of 47 ppb alongside a linear response from 47 ppb to 15 ppm was achieved using square wave voltammetry in addition to good selectivity. As a demonstration, the biosensor was applied to determine phenol concentrations in regional water samples from S. Korea.

Amperometric phenol biosensor based on covalent immobilization of tyrosinase onto an electrochemically prepared novel copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) film

An amperometric biosensor has been developed for the quantitative estimation of phenols in aqueous solution. The enzyme, tyrosinase, also called as poly phenol oxidase (PPO), was covalently immobilized on a novel copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) (PAPCP) film, electrochemically prepared onto an indium-tin-oxide (ITO) coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of phenol, at fixed bias voltage of −0.2 V versus Ag/AgCl in a phosphate buffer (pH 7.2). The electrode gives a wide linear response range of 1.35 –222.3, 1.6–118.8, 1.9–257.8 μM, for phenol, catechol and p-cresol, respectively. The response time is <10 s reaching to a 95% steady state current value, and 80% of the enzyme activity is retained for 4 months.

Microporous Gold Electrodes as Combined Biosensor/Electrochemical Detectors in Flowing Streams

Microporous gold electrodes are described for use as flow-through biosensor/electrochemical detectors in both flow injection analysis (FIA) as well as postcolumn detection in HPLC. Electrodes prepared by the covalent immobilization of tyrosinase onto the electrode surface (via a chemisorbed layer of thioctic acid and poly-lysine) are used to demonstrate the utility of the electrode. Such electrodes are shown to respond to a variety of substituted phenols under FIA conditions at applied potential of only −50 mV (vs. Ag/AgCl). Detection limits of 0.5 μM catechol and 3.4 μM phenol were achieved with a sample throughput rate of 38 samples/h. The electrode is shown to be stable for up to 14 days of use. The porous bioelectrode is shown to be useful as a postcolumn detector for substituted phenols following their separation by HPLC. Various techniques for immobilization of the enzyme onto the electrode surface (covalent and noncovalent) as well as factors affecting sensitivity and through-put (buffer ionic strength and addition of organic modifier) are discussed.