A new enzyme immobilization technique based on thionine-bovine serum albumin conjugate and gold colloidal nanoparticles for reagentless amperometric biosensor applications

A novel enzyme immobilization technique based on thionine-bovine serum albumin conjugate (Th-BSA) and gold colloidal nanoparticles (nano-Au) was developed. Thionine was covalently bound onto the BSA film with glutaraldehyde(GA) as cross-linker to achieve Th-BSA conjugate. The free amino groups of thionine were then used to attach nano-Au for the immobilization of horseradish peroxidase (HRP). Such nano-Au/Th-BSA matrix shows a favorable microenvironment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme. The proposed biosensor displays excellent catalytic activity and rapid response for H2O2. The linear range for the determination of H202 is from 4.9 x 10(-7) to 1.6 x 10(-3) M with a detection limit of 2.1 X 10(-7) M at 3 sigma and a Michaelies-Menten constant K-M(app) value of 0.023 mM.