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 detec tion elements in a competitive heterogeneous immunoassay employing either e lectrochemical 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, m ixed monolayers of a carboxylic acid-terminated thiol (thioctic acid) and a methyl-terminated thiol (butanethiol) were used to control the surface epi tope density. The microscopic structure of the resulting antibody and antig en arrays was characterized by AFM (atomic force microscopy), Individual, s urface-confined rabbit IgG antibodies could be directly imaged in contact m ode. The average height of the capture antibodies was found to be 7.1 nm; t he average antibody diameter, after correcting for tip convolution effects, was determined to be between 7 and 10 nm. The surface epitope density coul d be varied over approximately 2 orders of magnitude by changing the compos ition of the mixed monolayer, AFM was also used to characterize the antibod y-antigen binding characteristics of these immunosurfaces, and an average b inding efficiency of 22.8% was measured for rabbit IgG antibody arrays. In the second part of this study, the electrochemical detection scheme origina lly developed by Heineman and co-workers was adapted to our system. A calib ration data set was measured, and the linear least-squares correlation coef ficient (R-2) was found to be 0.993. Finally, the electrochemical and scann ing probe detection modes were directly compared. We find an excellent corr elation between the surface density of antibody-antigen complexes measured by AFM and the electrochemical response of the same immunosurfaces.