Dendrimer modified biochip for detection of 2,4,6 trinitrotoluene on SPR immunosensor: Fabrication and advantages

Abstract

This paper describes the fabrication and evaluation of new sensor chip technology based on application of polyamidoamine (PAMAM) dendrimer generation 4 (G4) for enhancing surface loading capacity and efficiency for selective and sensitive detection of explosive molecule trinitrotoluene (TNT) under competitive inhibition assay format. The fabrication process of a nano-scale biosensor chip surface was optimized at various steps involving thiol layer, cross-linker, dinitro phenylated-keyhole limpet hemocyanin (DNP-KLH) protein conjugate as ligand. The top ligand layer is supported by underlying 11-amino 1-undecanethiol hydrochloride (AUT) self-assembled monolayer (SAM) and PAMAM dendrimer G4. Bis-sulfosuccinimidyl suberate (BS 3) was used as homobifunctional crosslinker to react with amine groups at both the ends. PAMAM dendrimer activation was achieved over 11-amino 1-undecanethiol SAMs over gold chip. PAMAM sensor surfaces scored many advantages over the planer surfaces such as enhanced antibody immobilization, allowing the use of lower concentrated antibody to work under the regime of lower detection limit besides stable and robust surface leading to longer life. Bioactive thin films were fabricated over gold chip via layer-by-layer self-assembly methods. Biomolecular interaction between DNP-KLH conjugate surface and specific TNP-gly-KLH mouse IgG antibody was monitored through surface plasmon resonance (SPR) based real-time optical transducer. The quantitation of TNT on this bioactive surface was done using the competitive inhibition immunoassay. The DNP-KLH surface biosensor has shown a detection limit of 110 ppt for TNT molecule. This TNT specific biosensor holds the promise to be most sensitive, easy to regenerate, highly stable, low response loss sensor surface under competitive assay format. A 12 s injection of 10 mM glycine–HCl solution or 50 mM NaOH solution was found adequate for regeneration of DNP-KLH surface for repeated use. DNP-KLH sensor platform was checked for its repeatability and storability.