Electrochemical immunoassay of membrane P-glycoprotein by immobilization of cells on gold nanoparticles modified on a methoxysilyl-terminated butyrylchitosan matrix.
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 x 10(4) to 1.0 x 10(7) cells mL(-)(1) with a detection limit of 1.0 x 10(4) 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.
Du
Dan
D
Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry, Nanjing University, Nanjing 210093, PR China.
Ju
Huangxian
H
Zhang
Xueji
X
Chen
Jing
J
Cai
Jie
J
Chen
Hongyuan
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D018914:Cells, Immobilized / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods; D006801:Humans; D007118:Immunoassay; D020014:K562 Cells; D018625:Microscopy, Atomic Force
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies