Strategy to optimize the surface functionalization of immunosensors and improve their detection efficiency

• A strategy to improve the sensitivity of immunological sensors is provided. • The strategy is based on analyses of the different functionalization steps. • The chemical conditions have been optimized by examining the intermediate stages. • AFM, SEM and XPS provide information to increase the homogeneity and coverage. • The sensitivity (19%) and the limit of detection (16%) has been increased. The sensitivity of immunosensors depends on having effective procedures for functionalizing the transducer substrate and for immobilizing the recognition bioelement with the appropriate orientation. While important efforts have been devoted to develop processes able to induce highly oriented immobilization of the antibody, surface engineering to achieve a complete and homogeneous coverage of the transducer surface has received much less attention. Herein, we present an effective strategy to dissect each of the steps of substrate functionalization and thus propose improvements that result in enhanced sensitivity and limit of detection. The strategy is based on a systematic study using atomic force microscopy and X-ray photoelectron spectroscopy of the surfaces obtained in each of the intermediate stages of the functionalization process and, based on the results, propose changes in the chemical conditions for each of these stages that lead to an increase in homogeneity and degree of coverage. The proposed strategy has been successfully applied to an optical immunosensor based on anti-IL6. The proposed improvements have allowed an increase in sensitivity (19%) and a reduction in the limit of detection (16%.) Moreover, the approach can be easily extrapolated to any type of immunosensor based on the functionalization of transducer substrates with antibodies.