Electrochemical immunoassay based on a 96-well screen-printed ELISA plate

In this work, a novel electrochemical based-immunoassay for the specific detection of biomarkers employing a 96-well screen-printed microplate as the transducer surface, wasdeveloped. The capture element of the proposed sandwich immunoassay was efficiently immobilized onto the surface of the 96-well array and different concentrations of biomarkers, within a range of clinical interest, were assayed. The detection of the analyte of interest wasaccomplished by using alkaline phosphatase (AP) as the enzymatic label. The analytical signal was obtained by using two AP enzymatic substrates: 3-indoxyl phosphate/silver ions (3-IP/Ag+) [2] and the new hydroquinone diphosphate/silver ions (HQDP/Ag+), which results werecompared, namely in terms of sensitivity. In both cases, a biometallization process, where the enzymatic reaction gives rise to a compound that reduces silver ions in solution into a metallic deposit, took place. Thus, the silver enzymatically deposited on the electrode surface wasdetected through the oxidation peak of the silver when an anodic stripping scan was carried out. Moreover, different methodologies for the modification, in a reproducible manner, of this 96-well screen-printed electrochemical array, were studied. Therefore, the ELISA plate surface was modified with carbon nanotubes (CNTs), gold nanoparticles (GNPs) and with a hybrid conjugation of these carbon-metal nanomaterials (CNTs-GNPs). The reproducibility, stabilityand biofuncionality of these nanostructed transducer surfaces were also evaluated. The electrochemical behaviour of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding, limits of detection and reproducibility and the analytical figures of merit were obtained