Azurin immobilisation on thiol covered Au(111)

Abstract

Azurin is a blue single copper protein involved in the respiratory chain of denitrifying bacteria. The structural gene for azurin from Pseudomonas aeruginosa was cloned in an Escherichia coli recombinant strain. The protein overexpressed in the bacterial periplasmic space was subsequently purified. Two strategies were followed to anchor azurin to gold surfaces. First, the protein was immobilised on bare gold. Azurin adsorbs on gold ia its disulfide group. Scanning tunnelling microscopy (STM) inspection of the azurin–Au(111) interface revealed the formation of a closely packed protein monolayer and allowed individual azurin molecules to be resolved. In order to uncouple the protein layer from the metal, the gold surfaces were then covered with self-assembled monolayers of 11-mercaptoundecanoic acid. The changes in the sample morphology due to the protein adsorption have been investigated by atomic force microscopy (AFM). A fairly uniform distribution of protein molecules covers the surface. Owing to the tip broadening effect, an average protein diameter of about 20 nm was measured. An upper limit of 1 nN for the non-disruptive imaging force in the contact mode was found.