Study On Assay Methodologies For Tyrosinase Activity Based On Gold Nanoparticles

Tyrosinase plays an important role in the metabolism of animals and plants.The assay of tyrosinase activity provides not only the theoretical basis for clinical diagnosis and treatment, but also the basis to elucidate the pathogenesis of corresponding diseases,Meanwhile,it also has important applications in the fields of food preservation, environmental monitoring etc.Here we proposed two methods for tyrosinase activity detection based on biofunctionaliz- ed gold nanoparticles(AuNPs).Firstly, the experimental conditions of synthesis and modification of AuNPs were optimized. AuNPs were prepared following the traditional citrate reduction method. To obtain the concentrated AuNPs, centrifugation was adopted with the condition of 13000rpm×20min. The precipitate could be well dispersed in pH 7.0, 1mM,pHosphate buffer solution (PBS). The modification of the AuNPs with boronic acid proceeded in two steps. First, AuNPs were densely covered with a self-assembled monolayer of 16-mercaptohexadecanoic acid (MHDA).Then APBA was covalently linked to MHDA adsorbed on the surface of AuNPs in existence of N-(3-dimethylaminopropyl)-N-ethylcarbo-diimide hydrochloride (EDC) and the presence of N-hydroxysuccinimide (NHS). The obtained functionalized gold nanoparticles (APBA/MHDA/AuNPs) were dispersed in pH 7.2 HEPES buffer. The characteristic absorbance band of AuNPs in UV-vis spectrum shifts toλmax=530nm in APBA/MHDA/AuNPs,and a distinct absorption peak at the wavelength of 260nm indicates the successful linkage of APBA to the surface of AuNPs.A spectral method to analyze the activity of tyrosinase was then established by utilizing a specially designed substrate of tyrosinase,tyrosyltyrosine (Tyr-Tyr),which can be catalyzed to a briding product by the enzyme, subsequently triggers the aggregation of APBA/MHDA/AuNPs. The corresponding changes in UV-visible spectra were used to characterize the tyrosinase activity.The experimental conditions of tyrosinase activity assay were set as: the reacting system was in pH 6.5, 50mM PBS with the total volume of 0.1mL, the substrate concentration of 5mM,the reaction was carried out at 25℃for 30min,and terminated by heating up the system to 80℃. Afterward, 0.05mL of 5mM ascorbic acid was added to the system in order to convert dopaquinone to L-dopa. Finally 0.1mL of 9.5nM APBA/MHDA/AuNPs was added, and the mixture was incubated for 15min before UV-visible absorption spectroscopy was performed. The relationship between the absorbance in UV-visible spectra and the concentration of the enzyme was used to detect the activity of tyrosinase. The transmission electron microscope (TEM)images confirmed that the changes in UV-visible spectra was caused by the aggregation of APBA/MHDA/AuNPs triggered by link molecules.The detection range of this method is of 10-10~10-8U/mL.An electrochemical method to analyze the activity of tyrosinase was also proposed. In this method,another substrate of tyrosinase,tyramine was covalently modified onto the gold electrode surface through self-assembly and covalent cross-linking techniques.In the presence of tyrosinase, tyramine group on the electrode surface can be catalytically converted to dopa- mine, an o-hydroxy compound which can subsequently binding DNA/APBA/MHDA/AuNPs onto the electrode surface.Then,by using [Ru(NH3)5Cl]2+ as probe, which can adsorbed to the electrode surface by the electrostatic interaction between positively charged Ru and negatively charged DNA,the electrochemical signal can be detected to assay the activity of tyrosinase. The experimental conditions of tyrosinase activity assay were set as : the reaction system was in pH 6.5, 50mM PBS with the total volume of 0.1mL,catalysis was carried out at 25℃for certain time. The modified electrode was immersed in the solution of tyrosinase, which catalyzed the conversion of tyramine onto the electrode surface to dopaquinone. Subsequently, the electrode was immersed in 0.7% ascorbic acid solution for 30min in order to reduce dopaquinone to dopamine. Then the electrode was immersed in DNA/APBA/MHDA/AuNPs for 30min to allow the covalent binding of DNA/APBA/MHDA/AuNPs onto the electrode surface. Finally, by using [Ru(NH3)5Cl]2+ as probe, cyclic voltammetry (CV) was carried out, and the relationship between the peak currents in CVs and the concentration of tyrosinase was utilized to the detection. The proposed method can detect tyrosinase in the range of 10-14~10-10U/mL, which exhibits extremely high sensitivity as well as wide detection range.