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 °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 ∼33 pM, with negligible cross-reactivity against rabbit and chicken IgGs.