Abstract
An extensive study is described to identify the most suitable fluorescent label in magnetic microsphere sedimentation arrays. The investigated fluorescent labels, commonly used in multiplex analysis, include organic dyes, (fluorescein, Alexa488, Cy5) fluorescent proteins (R-Phycoerythrin, Allophycocyanine, PBXL-3) polymer nanoparticles (FluoSpheres, PD-Pt) and semiconductor nanocrystals (Quantum dots). DNA hybridization assays on magnetic microspheres were applied as model systems to reveal label performance. The fluorescent labels were characterized under optimized conditions regarding signal intensity, non-specific binding and photo-stability. The advantages and drawbacks of individual labels are discussed. The limit of detection and dynamic ranges are determined to compare the performance of selected labels. Detection limits of 2 × 10−10 mol/L are found for the determination of oligonucleotides using PBXl-3 as label, which is comparable with typical flow cytometer systems. The results and protocols are highly valuable for any type of bead based assays and can be easily transferred.
Similar content being viewed by others
Reference
Wilson R, Cossins AR, Spiller DG (2006) Encoded microcarriers for high-throughput multiplexed detection. Angew Chem Int Ed 45(37):6104–6117
Wentzell PD, Karakach TK (2005) DNA microarrays: is there a role for analytical chemistry? Analyst 130:1331–1336
Nagl S, Schaeferling M, Wolfbeis OS (2005) Fluorescence analysis in microarray technology. Microchim Acta 151:1–21
Yingyongnarongkul B et al (2003) Parallel and multiplexed bead-based assays and encoding strategies. Comb Chem High Throughput Screen 6:577–587
Moser C, Mayr T, Klimant I (2006) Microsphere sedimentation arrays for multiplexed bioanalytics. Anal Chim Acta 558(1–2):102–109
Mayr T, Moser C, Klimant I (2007) Luminescence decay time encoding of magnetic micro spheres. Anal Chim Acta 597:137–144
Glazer AN (1982) Phycobilisomes: structure and dynamics. Annu Rev Microbiol 36:173–198
Morseman JP et al (2000) Direct fluorescent detection of biotinylated oligonucleotides on glass slides using streptavidin labeled with PBXL-1 or phycoerythrin. Lumin Forum 6:4
Telford WG et al (2001) Cyanobacterial stabilized phycobilisomes as fluorochromes for extracellular antigen detection by flow cytometry. J Immunol Methods 254:13–30
Wang L et al (2005) Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays. Bioconjug Chem 16:194–199
Gerion D et al (2003) Room-temperature single-nucleotide polymorphism and multiallele DNA detection using fluorescent nanocrystals and microarrays. Anal Chem 75:4766–4772
Liang RQ et al (2005) An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe. Nucleic Acids Res 33:e17-1–e17/8
Haugland RP (2002) Handbook of fluorescent probes and research products, 9th edn. Molecular Probes, Eugene, OR, USA
Dubertret B et al (2002) In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298:1759–1762
Xu H (2003) Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res 31:e43-1–e43/10
Han M (2001) Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 19:631–635
Jaiswal JK et al (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol 21:47–51
Yang L, Tran D, Wang X (2001) BADGE, beadsArray for the detection of gene expression, a high-throughput diagnostic bioassay. Genome Res 11:1888–1898
Spiro A, Lowe M, Brown D (2000) A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Appl Environ Microbiol 66:4258–4265
Spiro A, Lowe M (2002) Quantitation of DNA sequences in environmental PCR products by a multiplexed, bead-based method. Appl Environ Microbiol 68:1010–1013
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mayr, T., Moser, C. & Klimant, I. Performance of Fluorescent Labels in Sedimentation Bead Arrays—A Comparison Study. J Fluoresc 19, 303–310 (2009). https://doi.org/10.1007/s10895-008-0416-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10895-008-0416-0