Electrochemical Immunoassay of Membrane P-glycoprotein by Immobilization of Cells on Gold Nanoparticles Modified on a Methoxysilyl-Terminated Butyrylchitosan Matrix

Abstract

A strategy to detect P-glycoprotein (P-gp) on cell membrane and quantify the cell number using electrochemical immunoassay was developed by effective surface immunoreactions and immobilization of cells on a highly hydrophilic interface, which was constructed by adsorption of colloidal gold nanoparticles on a methoxysilyl-terminated (Mos) butyrylchitosan modified glassy carbon electrode (Au-CS/GCE). Atomic force microscopy studies proved that the nanoparticles adsorbed on Mos-butyrylchitosan were efficient in preventing the cell leakage and retaining the activity of immobilized living K562/ADM leukemic cells. The incubation with P-gp monoclonal antibody and then the secondary alkaline phosphatase (AP) conjugated antibody introduced AP onto the K562/ADM cell immobilized on Au-CS/GCE. The bound AP led to an amperometric response of 1-naphthyl phosphate. Under optimal conditions the response was proportional to the logarithm of cell concentration in the range from 5.0 × 10⁴ to 1.0 × 10⁷ cells mL^-1 with a detection limit of 1.0 × 10⁴ cells mL^-1. The results were comparable to flow cytometric analysis of P-gp expression. This proposed method was practical, convenient, and significant in the clinic and cytobiology.