Fluorescence dye adsorption assay to quantify carboxyl groups on the surface of poly(methyl methacrylate) microbeads.

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.

[1]  R W Glenny,et al.  Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. , 1993, Journal of applied physiology.

[2]  P. Lebaron,et al.  Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment , 1998, Applied and Environmental Microbiology.

[3]  M. Kotaki,et al.  Surface modified nonwoven polysulphone (PSU) fiber mesh by electrospinning : a novel affinity membrane , 2006 .

[4]  Tiechuan Zuo,et al.  Micro Flow-through PCR in a PMMA Chip Fabricated by KrF Excimer Laser , 2005, Biomedical microdevices.

[5]  S. Dunbar,et al.  Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. , 2003, Journal of microbiological methods.

[6]  Da Xing,et al.  Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends , 2007, Nucleic acids research.

[7]  W. Göhde,et al.  Quantification of PCR products using microparticles and flow cytometry. , 2004, Annales de biologie clinique.

[8]  J. Butler.  Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. , 2000, Methods.

[9]  A. Gmitro,et al.  Assessment of acridine orange and SYTO 16 for in vivo imaging of the peritoneal tissues in mice , 2009, Journal of microscopy.

[10]  Steven A Soper,et al.  Photochemically patterned poly(methyl methacrylate) surfaces used in the fabrication of microanalytical devices. , 2005, The journal of physical chemistry. B.

[11]  Herwig Brunner,et al.  Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles. , 2004, Journal of nanoscience and nanotechnology.

[12]  Nan-Chyuan Tsai,et al.  SU-8 based continuous-flow RT-PCR bio-chips under high-precision temperature control. , 2006, Biosensors & bioelectronics.

[13]  Laurie Brown,et al.  Fabrication and characterization of poly(methylmethacrylate) microfluidic devices bonded using surface modifications and solvents. , 2006, Lab on a chip.

[14]  R. Giese.  Book reviewBioconjugate techniques: G.T. Hermanson, Academic Press, San Diego, New York, Boston, 1996, XXVI + 785 pp., price $99.00, ISBN 0-12-342335-X (hardback); $49.95, ISBN 0-12-342336-8 (paperback) , 1996 .

[15]  R. Faust,et al.  Synthesis, characterization, properties, and drug release of poly(alkyl methacrylate-b-isobutylene-b-alkyl methacrylate). , 2006, Biomacromolecules.

[16]  D. J. Mason,et al.  A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy , 1998, Applied and Environmental Microbiology.

[17]  S. Li,et al.  Enzyme immobilization onto micro-sized modified PMMA microspheres. , 2004, Methods and findings in experimental and clinical pharmacology.

[18]  B. Sandner,et al.  Die Medienabhängigkeit der alkalischen Hydrolyse von Methylmethacrylat‐Polymeren , 1983 .

[19]  Y. Ikada,et al.  Introduction of functional groups onto the surface of polyethylene for protein immobilization. , 1993, Biomaterials.

[20]  Y. Yamanashi,et al.  Surface Engineering of Polystyrene Dish for Improvement of Cell Adhesion Using Plasma Techniques , 2008 .

[21]  V. Fert,et al.  Simultaneous Detection of Multiplex-Amplified Human Immunodeficiency Virus Type 1 RNA, Hepatitis C Virus RNA, and Hepatitis B Virus DNA Using a Flow Cytometer Microsphere-Based Hybridization Assay , 2000, Journal of Clinical Microbiology.

[22]  P. Lebaron,et al.  Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems , 1998, Applied and Environmental Microbiology.

[23]  Frank D. Egitto,et al.  Plasma modification of polymer surfaces for adhesion improvement , 1994, IBM J. Res. Dev..

[24]  P. Bryant,et al.  A multiplex microsphere bead assay for comparative RNA expression analysis using flow cytometry. , 2004, Journal of biotechnology.

[25]  A. Schouten,et al.  Thin-Film Behavior of Poly(methyl methacrylates). 3. Epitaxial Crystallization in Thin Films of Isotactic Poly(methyl methacrylate) Using Crystalline Langmuir-Blodgett Layers , 1992 .

[26]  A. Sidoli,et al.  Production of a soluble and functional recombinant streptavidin in Escherichia coli. , 1998, Protein expression and purification.

[27]  Francis Barany,et al.  Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations. , 2005, Analytical biochemistry.

[28]  A. Wolff,et al.  Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature , 2007, Nucleic acids research.

[29]  H. Swaisgood,et al.  Analysis and optimization of methods using water‐soluble carbodiimide for immobilization of biochemicals to porous glass , 1982, Biotechnology and bioengineering.

[30]  Donald Wlodkowic,et al.  SYTO probes in the cytometry of tumor cell death , 2008, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[31]  B. Koopman,et al.  Effect of fluorochromes on bacterial surface properties and interaction with granular media , 1997, Applied and environmental microbiology.

[32]  D. Vignali.  Multiplexed particle-based flow cytometric assays. , 2000, Journal of immunological methods.

[33]  Martin Dufva,et al.  Fabrication of high quality microarrays. , 2005, Biomolecular engineering.

[34]  J. Vives-Rego,et al.  Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry , 1997, Applied and environmental microbiology.

[35]  K. P. Rao,et al.  Synthesis and characterization of poly(methyl methacrylate) functional microspheres , 2000 .