Preparation and characterization of bifunctional biopolymers for receptor-based liposomal immunosensing

In this study, we prepared bifunctional biopolymers for development of a novel liposomal immunosensing element. These biopolymers were produced such that a rat monoclonal antibody fragment Fab' was linked to a cardiac protein Troponin I (TnI) peptide by a cross-linking reagent, o-phenylenedimaleimide (o-PDM) or N-sucinimidyl 3-(2-pyridyldithio)propionate (SPDP). The biopolymer formation yields were approximately 10% for Fab-TnIMal and 30% for Fab-TnISPDP. Molar ratios of Fab' to SPDP or o-PDM and conjugated Fab' to TnI peptide and conjugation pH have considerable effects on the biopolymer yield. Purification of these biopolymers was achieved by employing size-exclusion HPLC. These biopolymers can bind to receptor channels on one end, while the peptide end can be recognized by an anti-TnI antibody serving as a protein linker to block the channels in the immunosensing element. Then reactions may be used where free analyte competes for cross-linker binding sites whereby channels are rendered active. Characterization of purified biopolymers was performed using gel electrophoresis, ELISAs, and a BIAcore instrument. Furthermore, results of real-time biospecific interaction experiments with use of the BIAcore show that competition binding reactions of free TnI peptide occurred in this new immunosensing design. The binding activities of these two biopolymers are slightly different.