Detection and analysis of phage M13KO7 using biosensor based on imaging ellipsometry

Antibody GP8 (anti-GP8) was used as a ligand to detect phage M13KO7 using a biosensor based on imaging ellipsometry (IE) without oriented ligand immobilization. When the phage M13KO7 in a sample interacted with the antibody in a microarray, a complex formed upon their affinity, and the layer covering the surface cell of the interaction became thicker. The variation in layer thickness was represented by gray-scale (or brightness) variation of the IE image. Direct and oriented immobilization of the antibody were analyzed to identify the best binding locations to capture the phage. In direct immobilization, the phage could be captured by anti-GP8 and not antibody GP3 (anti-GP3), which was attributed to different copy numbers of the coat proteins on the body of the phage specific to the two antibodies. This indicated that different binding locations could produce different detection efficiency. In oriented immobilization, the phage could be captured by the protein G-Fc-anti-GP3 model, with a low detection efficiency. In contrast, the avidin-biotin-based oriented immobilization could capture the phage with a high detection efficiency. Specifically, it could expose more Fab domains of anti-GP3 labeled with biotin; one avidin had four biotin-binding sites. To confirm detection and analyze binding location, the phage M13KO7 was imaged by transmission electron microscopy and atomic force microscopy. Our results suggest that binding location plays a significant role in the detection of big targets and contributes to enhancing biosensor detection sensitivity