Author(s): Wolter A, Niessner R, Seidel M, Wolter A, Niessner R, Seidel M
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Abstract Protein microarrays serve as measurement platforms for multianalytical applications. Small molecules, DNA, proteins, and cells are determined quantitatively. Amino-PEG surfaces can be a smart functional platform for protein microarrays with high signal-to-noise ratios. An effective step-by-step chemistry is developed for uniform presentation of terminal functional groups at each monolayer. Poly(ethelene glycol diamine) 2000 (DAPEG, 2000 g/mol) films were prepared onto silanized glass slides presenting epoxy groups. The uniformity of the grafted DAPEG monolayer is characterized by a chemiluminescence reaction using a chemiluminescence microarray reader with automated reagent supply and a horseradish peroxidase (HRP)/luminol reporter system. An intensity line plot on the horizontal axis was generated. The chemiluminescence intensities vary in a range of 2.6\%. Antibodies against HRP as model system were immobilized on N-hydroxysuccinimide activated DAPEG layers by means of a microcontact roboter system. Chemiluminescence signals of bound HRP are detected at each spot with a standard deviation of 2.9\%. The maximum antibody concentration that can be immobilized at the surface is determined with 1 mg/mL. Additives for an optimal spotting buffer are also studied. The use of the block-copolymer Pluronic F127 as antibody stabilizer is as well investigated as trehalose for the prevention of spot evaporation. The lowest detectable HRP concentration is 0.08 ng/mL determined on anti-HRP antibody microarrays. This study demonstrates how surfaces and analytical parameters for protein microarray applications can be characterized with a chemiluminescence readout system using a HRP reporter system.
This article was published in Anal Chem
and referenced in Journal of Bioprocessing & Biotechniques