Antibody-microarrays on hybrid polymeric thin film-coated slides for multiple-protein immunoassays

The development and characterization of protein microarrays fabricated on nanoengineered three-dimensional polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes in solutions through self-assembly process were described. Protein antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of the porous structure of the three-dimensional polymer film and thus, establishing a platform for parallel analysis. A method for fabrication of cytokines antibody-based protein microarray for simultaneous detection of multiple cytokines on the PET-coated slides was described. Cytokines play an important role in a wide range of physiological process, such as innate immunity, apoptosis, angiogenesis, cell growth, and differentiation. Therefore, simultaneous measurement of multiple cytokine expression levels is vital to reveal the complex cytokine network and to understand the development of certain human diseases. The protein microarray was printed by robotically spotting nine human cytokine and growth factor capture antibodies onto planar glass substrates. The fluoroimmunoassay of printed cytokine antibody microarrays were performed by incubating with cytokine samples, then binding by biotin-conjuated detection antibodies, and detecting by fluorophore conjugated streptavidin. This sandwich immunoassay-based protein microarrays protocol was developed for detection of multiple expression levels simultaneously with commercial available biotin-labeled detection antibody, so that no labeling of sera samples in required. This method was also optimized specifically for the special requirements of the cytokine detection, with special attention paid to selecting the surface chemistry of array substrate, array printing buffer and blocking buffer, and the fluorescent detection settings that yielded the highest sensitivity and selectivity against the lowest background. The dynamic ranges of the parallel assay for cytokines were around two to three orders of magnitude with limit of detection <10 pg/mL. This cytokine detection protein microarray system can be extended to a larger menu of cytokines and growth factors for applications such as profiling of cytokine expression.