COVALENT IMMOBILIZATION OF GLUCOSE OXIDASE ON WELL-DEFINED POLYMER-SILICON WAFER HYBRIDS VIA SURFACE-INITIATED RAFT-MEDIATED PROCESS

Abstract

A surface modification technique was developed for the functionalization of silicon surface with glucose oxidase (GOD). The silicon surface was first graft copolymerized with glycidyl methacrylate (GMA) via surface-initiated reversible addition-fragmentation chain-transfer (RAFT)-mediated process. GOD was then covalently immobilized through the ring-opening reaction between the amine groups of the GOD and the epoxide groups of the grafted GMA polymer chains. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface-modified surface after each modification stage. Increasing the thickness of the polymer layer and the immobilization time could allow a great amount of GOD to be immobilized on the silicon surface. The GOD-functionalized silicon hybrids are promising candidates for the silicon-based glucose biosensors. 1-4 It is of great importance to improve the biocompatibility of the silicon microdevices and to develop silicon surfaces with controllable cell adhesion. Immobilization of biomolecules on silicon substrates can be achieved via many methods such as adsorption, entrapment, covalent binding, etc. Functionalization of silicon surface with organics is a key in the development of new silicon-based biomedical devices. Tethering of polymer brushes on the solid substrate is an effective method of modifying the surface of the various substrates. 5-6 The dense polymer brushes can alter the properties of the surface, supply excellent mechanical and chemical protection to the substrate, and indicate new pathways to the functionalization of silicon surfaces for molecular recognition and sensing. 7-10 USA). The silicon wafers were cut into square chips of about 1.2 cm × 1.2 cm in size. The monomer, GMA, was passed through inhibitor-removing columns and then stored under an argon atmosphere at -10 °C. The initiator a,a'-azobis(isobutyronitrile) (AIBN, 97%) was obtained from Kanto Chemical Co. (Tokyo, Japan) and was recrystallized in anhydrous ethanol. The silane coupling agent (3-(trimethoxysilyl) propyl methacrylate (TMSPM, 98%)) was obtained from Aldrich Chemical Co. (St. Louis, MO, USA). The chain transfer agent (CAT), 1-phenylethyl dithiobenzoate (PDB), was synthesized according to the published procedures.19 GOD (Type II, 15500 units g-1 from Aspergillus niger) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's phosphate buffer solution (PBS, pH=7.4) was freshly prepared. Bio-Rad dye reagent for protein assay (Catalog No. 500-0006) was obtained from BioRad (Hercules, CA, USA). The other reagents were of analytical grade and were used without further purification unless otherwise mentioned. Immobilization process The process of surface functionalization of silicon was shown in Fig. 1. Coupling of the RAFT initiator to the silicon wafer was first achieved by adding TMSPM (0.41 mmol), AIBN (0.23 mmol), and PDB (0.46 mmol) at a molar ratio of 1.8:1:2 into 5 mL of dry DMF in a Pyrex tube containing the silicon chip. After purging the mixture with argon for 30 min, the surface coupling reaction continued at 60 °C for 24 h under an argon atmosphere. After the reaction, the modified substrate was washed with CH 2 Cl 2 and then with an ethanol/water mixture.