Sensitive immuno-detection using enzyme immobilized in nanoporous silica

Abstract

we developed a GOx immobilized nanoporous MCF silica and magnetic bead based sandwich immuno-detection system. Taking advantage of the unique structural characteristics of the nanoporous MCF silica, the large pore volume and surface area, and mesopores with large diameter and uniform sizes, we successfully immobilized the large enzyme, GOx into the pores using cross-linking agent, also modified the surface of the MCF silica with antibodies. As a result, the MCF-GOx-Ab was used to detecte antigens captured by magnetic bead-Ab through sandwich approach. At optimized conditions, 107beads/mL of magnetic beads, and 10min of catalytic reaction time, using GOx immobilized in MCF silica it could detect model antigens, human IgG from 100pM to 100nM with less nonspecific binding to chicken IgG., and it showed about 20times higher sensitivity than conventional immunoassay using Ab-GOx conjugate antibodies made in our lab. To improve the sensitivity of this immuno detection, several factors still need to be optimized, for instance, the concentration of magnetic beads, the enzyme loading percentage, the quantity of antibodies linked to the surface of MCF silica and the concentrations of the substrates for catalytic reaction. Also different enzymes like HRP (horseradish peroxidase), or ALP (alkaline phosphatase) could be tried to develop a sensitive immunoprobe. As a new immunosensing technology, it must have profound potential not only in detection of various antigens but pathogens as well.