Synthesis and characterization of hapten-quantum dots bioconjugates: Application to development of a melamine fluorescentimmunoassay

Abstract

A general and universal analytical strategy for characterization of hapten-BSA conjugates based on complementary optical spectroscopy and molecular mass spectrometry techniques is here described. The proposed procedure provides highly-valuable information about the molecular weight of the conjugate, its stoichiometry and the concentration of the precursors (hapten and BSA) in the conjugate; such information is of great analytical interest for further development of novel quantitative immunoassays. Further, due to great demand of new, simple and robust methodologies for the melamine analysis in milk infant formula, a new immunoprobe melamine-bovine serum albumin-quantum dot was synthetized, characterized and successfully applied in a competitive fluorescent quantum dot-based immunoassay. It should be highlighted that the limit of detection achieved without any sample pretreatment, 0.15 mg kg(-1) for melamine in milk infant formula, is one order of magnitude lower than the maximum concentration level allowed by international legislation in such type of samples. Finally, this simple approach was validated by the use of an alternative technique (HPLC-UV) for the analysis of melamine in contaminated milk infant formula, showing a good agreement between the results obtained by using both analytical methodologies.