Tyrosinase immobilization on functionalized porous silicon surface for optical monitoring of pyrocatechol

Abstract

Immobilized Tyrosinase enzyme was widely investigated for developing sensitive and specific biosensors for determination of phenolic compounds. However, the performance and stability of these biosensors largely depend upon the used support material and method of enzyme immobilization. In this work, covalent and stable attachment of active Tyrosinase on porous silicon (PSi) support for the detection of phenolic derivatives was achieved. A multistep functionalization strategy was developed in mild conditions allowing the activity retention of the immobilized enzyme. The surface was characterized with contact angle measurements, SEM microscopy and FTIR spectroscopy. The results confirmed the attachment of Tyrosinase enzyme on the functionalized PSi surface. The enzymatic activity of immobilized Tyrosinase and the optical detection of pyrocatechol were studied basing on the assessment of the UV/Vis absorbance at 500 nm of a colored azo-dye product formed from the reaction of 3-methyl-2-benothiazolinone hydrazone (MBTH) reagent with the Quinone issuing from the enzymatic oxidation of pyrocatechol. The surface concentration of active Tyrosinase was estimated to be Γ = 1.135 × 1015 molecules covering the whole PSi surface. The calibration curve of pyrocatechol detection shows linearity over a wide concentration range (1–100 μM) with a correlation coefficient of 0.995 and a detection limit of 0.43 μM. These results show the efficiency of the Tyrosinase-modified PSi surface for phenol detection and the possibility to use the structure in real applications.