Development and molecular recognition of Calixcrownchip as an electrochemical ALT immunosensor

The developments of immunosensors with a variety of formats are increasingly finding applications in clinical diagnostics and biological researches. A strategy for the immunoassay and preparation of Calixcrownchips is proposed. This strategy is based on the immobilization of antigens or antibodies on the surface of Calixcrown and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody or antigen, with its activity determined by using tetramethylbenzidine (TMB) as an electrochemical substrate. The present study includes general considerations of the competitive immunoreaction protocols. Alanine aminotransferase (ALT) monoclonal antibody (anti-ALT-mAb) was successfully immobilized on thiol derivative of Calixcrown fixed to a gold surface. ALT antigen was detected by competitive immunoreactions based on microarrays of anti-ALT-mAb or antigen immobilized on the surface of the Calixcrownchip. For the anti-ALT-mAb immobilized microarray the dynamic range is 0.05 ng/mL-10 mu g/mL, the detection limit is 0.05 ng/mL and the sensitivity is 10 nA/(ng/mL) respectively. The Calixcrownchip immunosensor microarray provided much better technical performance than a comparable enzyme sensor with immobilized-anti-ALT-mAb. To investigate the complexation site, the structures of the complexes formed between the crown-5-ether moiety of Calix[4]arene and protonated Arginine and Lysine were determined by minimizing the complex formation energies. The complex stability depends on the number of amine groups in the alkyl chain of the amino acid and also the number of methylene groups between the amine groups of the amino acid.