Controlled antibody orientation on Fe3O4 nanoparticles and CdTe quantum dots enhanced sensitivity of a sandwich-structured electrogenerated chemiluminescence immunosensor for the determination of human serum albumin

Abstract

• Impact of antibody orientation in the performance of ECL is firstly investigated. • Protein G results antibody orientation and blocks nonspecific binding sites simultaneously. • Photoluminescence intensity was 11.8 times higher with full antibody orientation than without. • ECL intensity was almost nine times higher with full antibody orientation than without. In the present study, the sensitivity of an electrochemiluminescence (ECL) immunosensor was enhanced by controlled antibody orientation, developed by modification of both Fe 3 O 4 nanoparticles (FNs) and CdTe quantum dots (QDs) as nanocarriers and luminance labels, respectively. Magnetic FNs were selected as nanocarriers for easy magnetic separation and were coated with SiO 2 layer for immobilization of primary antibodies (Ab1). Protein G on the FN surface could support Ab1 with a high degree of controlled orientation and could block nonspecific binding sites to enhance the sensitivity and selectivity of the ECL immunosensor. Moreover, CdTe QDs with protein G coatings were selected as signal labels and were conjugated with secondary antibodies with a high degree of orientation. The sandwich-structured immunosensor exhibited remarkable stability according to cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Photoluminescence and ECL intensities of the developed immunosensor were 11.8 and 9 times higher with controlled antibody orientation than without it. The developed immunosensor, used to quantify human serum albumin (HSA), exhibited a wide linear range (20–680 ng/mL), and a detection limit of 12 ng/mL. Furthermore, HSA-spiked serum samples were used to evaluate the accuracy and reliability of the immunosensor. The results indicate that antibody orientation substantially improves the sensitivity of sandwich-structured ECL immunosensors for clinical bioassay.