High performance immunoassay using immobilized enzyme in nanoporous carbon.
A highly stable immunoassay format was constructed using signal-generating enzyme immobilized in nanoporous carbon. A mesocellular carbon foam, called MSU-F-C, was loaded with horseradish peroxidase (HRP), followed by cross-linking of the enzyme using glutaraldehyde (GA) and modification of the surface with anti-human IgG through EDC/sulfo-NHS chemistry. The resulting MSU-F-C/HRP/anti-human IgG stably retained immobilized enzymes and antibodies, showing higher thermal stability. The MSU-F-C/HRP/anti-human IgG retained about 80 % of initial enzyme activity at 40 degrees C after a 5 h incubation, while the HRP/anti-human IgG conjugate resulted in almost 90% loss of initial activity in the same condition. In bead-based immunoassays, the signal amplification using MSU-F-C/HRP/anti-human IgG enabled the sensitive colorimetric detection of a target analyte, human IgG, in a detection limit of approximately 33 pM, with negligible cross-reactivity against rabbit and chicken IgGs.
Piao
Yunxian
Y
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
Lee
Dohoon
D
Kim
Jungbae
J
Kim
Jaeyun
J
Hyeon
Taeghwan
T
Kim
Hak-Sung
HS
D000818:Animals; D000906:Antibodies; D002244:Carbon; D002645:Chickens; D004800:Enzymes, Immobilized; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D008280:Magnetics; D036103:Nanotechnology; D011817:Rabbits; D012680:Sensitivity and Specificity
D000906:Antibodies; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D002244:Carbon; D006735:Horseradish Peroxidase