Comparative Assessment of Affinity-Based Techniques for Oriented Antibody Immobilization towards Immunosensor Performance Optimization

Immunosensor sensitivity and stability depend on a number of parameters such as the orientation, the surface density, and the antigen-binding efficiency of antibodies following their immobilization onto functionalized surfaces. A number of techniques have been developed to improve the performance of an immunosensor that targets one or both of the parameters mentioned above. Herein, two widely employed techniques are compared for the first time, which do not require any complex engineering of neither the antibodies nor the surfaces onto which the former get immobilized. To optimize the different surface functionalization protocols and compare their efficiency, a model antibody-antigen system was employed that resembles the complex matrices immunosensors are frequently faced with in real conditions. The obtained results reveal that protein A/G is much more efficient in increasing antibody loading onto the surfaces in comparison to boronate ester chemistry. Despite the fact, therefore, that both contribute towards the orientation-specific immobilization of antibodies and hence enhance their antigen-binding efficiency, it is the increased antibody surface density attained with the use of protein A/G that plays a critical role in achieving maximal antigen recognition.