High performance immunoassay using immobilized enzyme in nanoporous carbon.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
The Analyst (Impact Factor: 4.23). 06/2009; 134(5):926-32. DOI: 10.1039/b813451k
Source: PubMed

ABSTRACT 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.

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