An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification.
A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10(-14) to 10(-5) gml(-1) was acquired with a relatively low detection limit of 3.5 x 10(-5) gml(-1) for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.
Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China.
Yali
Y
Yuan
Ruo
R
Yuan
Yaqin
Y
Chai
Ying
Y
Zhuo
Lijuan
L
Bai
Yuhong
Y
Liao
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000627:Aminophenols / Q000737:chemistry; D000814:Aniline Compounds / Q000737:chemistry; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry; D009245:NADH Dehydrogenase / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D013379:Substrate Specificity
D000627:Aminophenols; D000814:Aniline Compounds; D007074:Immunoglobulin G; D009943:Organophosphorus Compounds; D001710:Biotin; C068798:4-aminophenylphosphate; D019809:Streptavidin; D009245:NADH Dehydrogenase; D000469:Alkaline Phosphatase; C026729:4-aminophenol