Heterogeneous Immunosensing Using Antigen and Antibody Monolayers on Gold Surfaces with Electrochemical and Scanning Probe Detection

We report the use of antibody and antigen monolayer immunosurfaces as detection elements in a competitive heterogeneous immunoassay employing either electrochemical or scanning probe detection. Antibody or antigen monolayers were prepared by covalent attachment of the desired immunoreagent to a two-component self-assembled monolayer via amide linkages. More specifically, mixed monolayers of a carboxylic acid-terminated thiol (thioctic acid) and a methyl-terminated thiol (butanethiol) were used to control the surface epitope density. The microscopic structure of the resulting antibody and antigen arrays was characterized by AFM (atomic force microscopy). Individual, surface-confined rabbit IgG antibodies could be directly imaged in contact mode. The average height of the capture antibodies was found to be 7.1 nm; the average antibody diameter, after correcting for tip convolution effects, was determined to be between 7 and 10 nm. The surface epitope density could be varied over approximately 2 orders of magnitude by changing the composition of the mixed monolayer. AFM was also used to characterize the antibody−antigen binding characteristics of these immunosurfaces, and an average binding efficiency of 22.8% was measured for rabbit IgG antibody arrays. In the second part of this study, the electrochemical detection scheme originally developed by Heineman and co-workers was adapted to our system. A calibration data set was measured, and the linear least-squares correlation coefficient (R2) was found to be 0.993. Finally, the electrochemical and scanning probe detection modes were directly compared. We find an excellent correlation between the surface density of antibody−antigen complexes measured by AFM and the electrochemical response of the same immunosurfaces.