title: Preparation and characterization of functional poly(ethylene glycol) surfaces for the use of antibody microarrays.
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.
authors.affiliation: Institute of Hydrochemistry, Technische Universität München, D-81377 München, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Wolter
authors.forename: Reinhard
authors.initials: R
authors.lastname: Niessner
authors.forename: Michael
authors.initials: M
authors.lastname: Seidel
mesh_terms: D000588:Amines / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D002477:Cells / Q000473:pathology; D004247:DNA / Q000032:analysis; D004800:Enzymes, Immobilized; D005898:Glass / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D020442:Poloxamer / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry; D013388:Succinimides / Q000494:pharmacology; D013499:Surface Properties
chemical_list: D000588:Amines; D000906:Antibodies; D004800:Enzymes, Immobilized; D011506:Proteins; D012821:Silanes; D013388:Succinimides; D020442:Poloxamer; D011092:Polyethylene Glycols; D008165:Luminol; D004247:DNA; D006735:Horseradish Peroxidase; C001426:N-hydroxysuccinimide