Highly sensitive phenol biosensor was developed by using well-dispersed carbon nanotubes (CNTs) in enzyme solution and adding CNTs in enzyme electrodes. First, the intact CNTs were dispersed in aqueous tyrosinase (TYR) solution, and TYR molecules were precipitated and crosslinked to prepare the sample of enzyme adsorption, precipitation and crosslinking (EAPC). EAPC exhibited 10.5- and 5.4-fold higher TYR activity per mg of CNTs as compared to enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC), respectively. EAPC retained 29% of its initial activity after incubation at 40 °C for 128 h, while EA and EAC showed no residual activities, respectively. In biosensing a model phenolic compound of catechol, the sensitivities of EA, EAC and EAPC electrodes on glassy carbon electrode (GCE) were 34, 281 and 675 µA/mM/cm2, respectively. When 90 w/w% CNTs were added to the enzyme electrodes, the sensitivities of EA, EAC, and EAPC electrodes were 146, 427, and 1160 µA/mM/cm2, respectively, and the EAPC electrode showed a 2.3-fold increase in sensitivity upon CNT addition. Catechol and phenol could also be detected by EAPC on the screen-printed electrode (SPE), with sensitivities of 1340 and 1170 µA/mM/cm2, respectively. The sensitivity of EAPC-SPE for phenol detection in the effluent from real municipal wastewater treatment plant was 1100 µA/mM/cm2. The sensitivity of EAPC-SPE retained 74% of its initial sensitivity after incubation at 40 °C for 12 h. The combination of EAPC immobilization and CNT addition has great potential for application in the development of sensitive enzyme biosensors for various analytes and phenols in water environments.
Keywords: Carbon nanotubes; Enzymatic biosensor; Enzyme adsorption, precipitation and crosslinking; Phenolic compounds, Screen-printed electrode; Tyrosinase.
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