Dual immunoassay of human chorionic gonadotropin and human placental lactogen at a miocrofabricated substrate by scanning electrochemical microscopy

A dual imtnunoassay of human chorionic gonadotropin (HCG) and human placental lactogen (HPL) at a microfabricated substrate was studied using scanning electrochemical microscopy (SECM). Anti-HCG and anti-HPL were microspotted on 50 × 50 μm² raised flat squares separated from each other by 150 μm at a glass substrate. A 10 μ1 sample solution containing HCG and/or HPL was then dropped onto the substrate followed by treatment with a solution of horseradish peroxidase (HRP)-labeled anti-HCG and HRP-labeled anti-HPL. The SECM measurements were carried out at 0.05V vs. Ag|AgCl in the presence of ferrocenylmethanol (FMA) and H₂O₂ to detect FMA⁺ generated by the HRP reaction. The SECM images of the substrates showed clearly the large response areas which corresponded to the squares with HCG and/or HPL. The detection limits were 0.1 IU ml^-1 for HCG and 3 ng ml^-1 for HPL.