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3D-Microstructured Protein Chip for Cancer Diagnosis

Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients’ and 28 healthy donors’ sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.