earticle

영어

We demonstrate here a highly-sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered crosslinked TYR-MNP aggregates (M-EC-CNT). M-EC-CNT was highly loaded with TYR and MNP, allowing for high activity and quick magnetic capture, and the good TYR stability could be achieved due to the multiple covalent linkages on the surface of TYR1,2).
Magnetically-capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet for an electrochemical assay. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was recycled to catechol for substrate recycling. The combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng-1 mL-1 and a detection limit of 0.19 ng mL-1 (1.2 pM), which represent better performance than any other electrochemical immunosensors based on TYR-ALP for substrate recycling. The present approach also displayed a long-term stability by showing negligible loss of electrochemical signal after the reagents were stored in an aqueous buffer at 4ºC for more than 6 months. High sensitivity and stability of the present immunosensor seem to result from stabilization of multi-layered enzyme assembly on conductive CNTs. We anticipate that the developed immunosensing system meets the current demanding in high performance electrochemical immunoassay, in terms of sensitivity, stability and reproducibility, and might be applied to detection of a variety of analytes.