Electrochemical Polymerization of (2-Dodecyl-4,7-di(thiophen-2-yl)-2H-benzo[d][1,2,3] triazole): A Novel Matrix for Biomolecule Immobilization

A recently synthesized conducting polymer [poly(2-dodecyl-4,7-di(thiophen-2-yl)-2H-benzo[d][1,2,3] triazole (PTBT)] was tested as a platform for biomolecule immobilization. After electrochemical polymerization of the monomer (TBT) on graphite electrodes, immobilization of glucose oxidase (GOx, beta-D-glucose: oxygen-1-oxidoreductase, EC 1.1.3.4) was carried out. To improve the interactions between the enzyme and hydrophobic alkyl chain on the polymeric structure, GOx and isoleucine (Ile) amino acid were mixed in sodium phosphate buffer (pH 7.0) with a high ionic strength (250 x 10(-3) M). The solution is then casted on the polymer film, and the amino groups in the protein structure were crosslinked using glutaraldehyde (GA) as the bifunctional agent. Finally, the surface was covered with a perm-selective membrane. Consequently, cross-linked enzyme crystal (CLEC) like assembles with regular shapes were observed after immobilization. Microscopic techniques such as scanning electron microscopy (SEM) and fluorescence microscopy were used to monitor the surface morphologies of both the polymer and the bioactive layer. Electrochemical responses of the enzyme electrodes were measured by monitoring O-2 consumption in the presence of glucose at -0.7 V. The optimized biosensor showed a very good linearity between 0.05 and 2.5 x 10(-3) M with a 52 s response time and a detection limit (LOD) of 0.029 x 10(-3) M to glucose. Also, kinetic parameters, operational and storage stabilities were determined. K-m and I-max values were found as 4.6 x 10(-3) M and 2.49 mu A, respectively. It was also shown that no activity was lost during operational and storage conditions. Finally, proposed system was applied for glucose biomonitoring during fermentation in yeast culture where HPLC was used as the reference method to verify the data obtained by the proposed biosensor.