title: Fluorescence dye adsorption assay to quantify carboxyl groups on the surface of poly(methyl methacrylate) microbeads.
abstract: Microbead-based assays have evolved into powerful tools for the multiplex detection of biomolecules. Analytes are captured by DNA or protein capture molecules which are coupled on microbead surfaces. A homogeneous carboxylation of microbeads is essential for the optimal and reproducible coupling of capture molecules and thus a prerequisite for an optimal multiplex microbead-based assay performance. We developed a simple fluorescence dye adsorption assay for the description of microbead carboxylation and for the prediction of coupling successes of capture molecules. Using the fluorescence dye SYTO-62 it is possible to quantify the degree of carboxylation of poly(methyl methacrylate) (PMMA) microbeads within 1 h in a multiplex format by fluorescence microscopy or flow cytometry. Compared to conventional bulk assays which only provide an average degree of carboxylation the main advantage of the SYTO-62 assay is the single microbead analysis and therefore the description of the qualitative distribution of carboxylation in microbead populations. The SYTO-62 assay is sensitive enough to even determine weak carboxylation. Also, the quality of microbeads can be evaluated. To our knowledge this is the first report which applies a reversible noncovalent fluorescent dye adsorption assay to quantify the degree of carboxylation on surfaces.
authors.affiliation: Department of Biology, Chemistry and Process Technology, University of Applied Sciences, Senftenberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rödiger
authors.forename: Mirko
authors.initials: M
authors.lastname: Ruhland
authors.forename: Carsten
authors.initials: C
authors.lastname: Schmidt
authors.forename: Christian
authors.initials: C
authors.lastname: Schröder
authors.forename: Kai
authors.initials: K
authors.lastname: Grossmann
authors.forename: Alexander
authors.initials: A
authors.lastname: Böhm
authors.forename: Jörg
authors.initials: J
authors.lastname: Nitschke
authors.forename: Ingo
authors.initials: I
authors.lastname: Berger
authors.forename: Ingolf
authors.initials: I
authors.lastname: Schimke
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
mesh_terms: D000327:Adsorption; D002245:Carbon Dioxide / Q000032:analysis* / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D015345:Oligonucleotide Probes / Q000737:chemistry; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D011786:Quality Control; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D002245:Carbon Dioxide; C041069:carboxyl radical; D019904:Polymethyl Methacrylate; D019809:Streptavidin