An ellipsometric study on the density and functionality of antibody layers immobilized by a randomly covalent method and a protein a-oriented method

Ellipsometry is used to quantitatively evaluate the antigen-sensing capability of immuno-surfaces. The ellipsometric measurement is a rapid, label-free, and in situ detection; however, quantification of analytes by ellipsometry is not unambiguous. The proper use of the effective thickness as a measure for the amount of analytes is addressed in this article. Taking the effective thickness as the quantification reference, we find that employing protein A to immobilize antibodies makes the antigen capture two to three times higher than the covalent immobilization technique using N-succinimidyl 4-maleimidobutyrate (GMBS). Besides, the results also suggest a uniform orientation of antibodies achieved by protein A. Increasing the density of surface antibodies would improve the sensitivity as well; however, the effect is more crucial for the protein A immuno-surface and appears to be less important for the GMBS immuno-surface. Therefore, the optimal immobilization strategy should include an orientation layer, such as protein A, and maximize the amount of oriented antibodies on the surface to further improve the efficiency for antigen detection.