Effects of different self-assembled monolayers on enzyme immobilization procedures in peroxidase-based biosensor development

Different immobilization procedures for horseradish peroxidase (HRP) were investigated using, as support matrices, self-assembled monolayers (SAM) formed on gold electrodes. The influences of these immobilization processes in the biosensor performance were also evaluated. For this, monolayers were prepared from thiols with different structures, carbon chain sizes and terminal groups. It was shown that the thiol carbon chain size influences especially in monolayer coverage and, consequently, in the biomolecule immobilization efficiency. From the studies carried out for SAM characterization on the electrode surface it was verified that thiols with shorter chains (n < 9) tend to form monolayers with a considerable amount of defects on the gold surface that lead to a lower coverage. However, thiols with a longer carbon chain present a higher coverage degree, which are not suited as substrate to the development of electrochemical biosensors, because they passivate the transduction interface, making difficult the electron transfer and, consequently, reducing electrode sensitivity. In relation to the enzyme immobilization on gold electrodes, it was verified, using different techniques, that monolayers that possess –NH2 terminal groups provided the best results, probably due to the use of glutaraldeyde as ligand during the immobilization process. Analyzing biosensor performances for hydrogen peroxide, it was verified that SAM formed by cysteamine is the most adequate for HRP immobilization, because it provides better efficiency for enzyme immobilization associated to high sensitivity for H2O2.