Preparation and characterization of functional poly(ethylene glycol) surfaces for the use of antibody microarrays.
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.
Wolter
Anne
A
Institute of Hydrochemistry, Technische Universität München, D-81377 München, Germany.
Niessner
Reinhard
R
Seidel
Michael
M
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
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