Micro and nanotechnology for early diagnosis and detection of rheumatic diseases-molecular markers

Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids contributes to the diagnosis of rheumatic diseases associated with rheumatoid factor. Here, the proinflammatory cytokine Tumor Necrosis Factor alpha (TNF-?) was chosen as the first target of interest. We report on the development of a fast sandwich immunosorbent assay: anti-TNF-? antibody (Ab) immobilization on a pre-activated 3D micro-structured glass slide, coupled with direct fluorescence readout method. Two differently functionalized glass - Ab-immobilization substrates - were compared: O2 PLASMA/APTES activated glass - having a two-dimensional structure-, and the polystyrene (PS) beads coated glass, -having a 3D structure-. The 3D structured substrate was aimed to increase antibody anchorage sites. Then, the optimization of buffer media was carefully investigated with respect to non-specific protein binding. As a first step towards real sample analysis, a proof of principle of on/ off cytokine detection has been analyzed in the presence of human serum. High-density one-step immobilization of anti-TNF-? antibody onto nanobead-coated glass slide chip has been demonstrated to be a promising device for application in in vitro diagnostic and profiling of cytokines with potential application toward personalized therapeutic interventions.