Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins. These parameters relate to surface chemistry and to immobilization conditions (printing buffers, washing etc.). In this study, we have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions were evaluated for the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that the biological activities of the immobilized antibodies were dependent on the surface chemistry and on the immobilization procedure. Optimal immobilization conditions were different for each antibody. Limit of detection in tumor antigen as low as 10 pM can reach under optimized conditions. Our 3D microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody-antigen system on the same chip. © 2011 Elsevier B.V. All rights reserved.