Imaging of immobilized antibody layers with scanning electrochemical microscopy

Visualization of immobilized antibodies can be achieved with scanning electrochemical microscopy (SECM) by saturation of the antigen binding sites with an alkaline phosphatase-antigen conjugate, which catalyzes hydrolysis of the redox-inactive 4-aminophenyl phosphate to the redox-active 4-aminophenol (PAP). PAP was detected in the collection mode at an amperometric SECM tip. The tip current reflects the density of active binding sites in the immobilized antibody layer. The application of this approach for immunosensing research has been demonstrated with the optimization of a covalent immobilization procedure of antibodies on glass. The special advantages and present limitations of the procedures are discussed.