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Gold nanoparticles (GNPs) conjugated with biomolecules are promising building blocks for assembly into nanostructured functional materials for developing biomarker platforms because of their size dependent optical and electrical properties. Biocompatible GNPs were synthesized using glutamic acid as a reducing agent and the interaction between bovine serum albumin (BSA) and GNPs was investigated using fluorescence and circular dichroism (CD) spectroscopies. The binding constant (Kb) of protein (BSA) to GNPs was determined by measuring the quenching of the fluorescence intensity of tryptophan residues of the protein molecules after conjugation. The conformational change in BSA at its native form after conjugation with GNPs confirmed that protein undergoes a more flexible conformational state on the boundary surface of GNPs after bioconjugation. The CD studies further showed a decrease in the α-helical content after conjugation. The results confirmed that the change in conformation was larger at higher concentrations of GNPs.

For the generation of antibodies against small hapten molecules, the hapten is cross-linked with some carrier protein to make it immunogenic. However, the formation of such conjugates is not always reproducible. This may lead to inconsistent hapten−protein stoichiometries, resulting in large variations in the generation of the desired antibodies. In the study described here the hapten (mercaptopropionic acid derivative of atrazine) was coupled to carrier protein at five different molar ratios. The hapten−protein conjugates prepared were characterized thoroughly by spectrophotometric absorption, fluorescence, matrix-assisted laser desorption ionization (MALDI), and gel electrophoresis methods, before being used for the immunization and assay purposes. Electrophoresis and fluorescence methods were very useful in detecting hapten−protein cross-linking while MALDI-MS and spectrophotometric detection provided qualitatively comparable hapten density. The production of specific antibodies was sought following the generation of appropriate hapten−protein conjugates. A high antibody titer with moderate antibody specificity was obtained with hapten density around 15 molecules per carrier protein. The study proved useful for monitoring the course of hapten−protein conjugation for the production of specific antibodies against small molecules.

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