Resorc[4]arene-based site directed immobilization of antibodies for immunosensors development

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibody to the surface of the sensor. In fact, antibodies need to be immobilized with a high density and good orientation to allow the easy detection of antigens. The influence of nonspecific bindings should be minimized to improve the detection performance. Most of immobilizing methods lead to randomly oriented antibodies on the surface, which results in a low density of binding sites and alleviation of immunoaffinity of the antibodies. Therefore, oriented immobilization is required for the improvement of the performance enhancement. Calix[4]arene derivatives have been proposed as an alternative tool for the oriented immobilization of antibodies thanks to their unique three‐dimensional surface, which can be functionalized at both the upper and lower rims with several functional groups.1 Within the calixarene family, resorcinol‐derived cycloligomers, namely resorcarenes, behave as abiotic artificial receptors having enforced cavities of molecular dimension. 2 To ensure the orientation control of antibodies on the sensor surface, we synthesized several resorc[4]arene derivatives able to self-assemble onto gold surface thanks to the thiol groups present on their structure.3 After the spectroscopic characterization of resorc[4]arene self‐assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab‐Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole–dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, the bis-crown resorcarene showed a major binding force towards Ab‐Ins thanks to the H‐bond interactions with the amine protein groups. Based on these findings, the resorcarene‐based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development. Acknowledgements The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. This manuscript is dedicated to the memory of Professor Maurizio Botta (University of Siena, Department of Biotechnology, Chemistry and Pharmacy).