Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.