Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection

Microarrays

Ingeborg Hospach²  Yvonne Joseph¹  Michaela Kathrin Mai²  Nadejda Krasteva²  Gabriele Nelles² 

[1] Institute of Electronic and Sensor Materials, Technische Universität Bergakademie Freiberg, Gustav-Zeuner-Strasse 3, 09599 Freiberg, Germany; E-Mail:;Materials Science Laboratory, Sony Deutschland GmbH, Hedelfinger Strasse 61, 70327 Stuttgart, Germany; E-Mails:

关键词: microarray;    protein;    antibody;    cytokines;    spotting;    hydrogel;    biosensor;    fluorescence readout;   

DOI  :  10.3390/microarrays3040282

来源: mdpi

【 摘 要 】

Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.