Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 213

functional groups to enable covalent coupling. Song et al. [ 58 ] used a similar
method to encapsulate a phosphorescent platinum(II) porphyrin (PtTFPP) into a
copolymer of vinyl chloride, ethyl acetate and maleic acid. The monodispersed
beads (40

300 nm depending on solvent and precipitation speed) were
found to be stable against aggregation and be redispersible after centrifugation,
which was not the case for poly(vinyl chloride) beads used for comparison.
Moreover, very high dye loading (up to 3.3% w/w) enabled preparation of brighter
beads. Unfortunately, the degree of oxygen quenching in the beads remains unclear.
The particles have been successfully used to covalently tag a C-reactive protein
(CRP) monoclonal antibody and provide a very sensitive time-resolved phospho-
rescent assay for CRP. We recently reported nanobeads based on poly(methyl
methacrylate- co -methacrylic acid) [ 17 ] with an average diameter of 55 nm. Lumi-
nescence of the iridium(III) coumarin complex incorporated into the beads was
affected only in a minor way by oxygen. The beads may be suitable for labeling
purposes due to the presence of reactive carboxyl groups on the surface.
Lanthanide complexes are particularly attractive in such applications since their
luminescence is only minimally quenched by oxygen and, therefore, the choice is
not limited by gas-blocking polymers. Thus, several highly sensitive lanthanide-
based immunoassays were reported recently. For example, Huhtinen et al. [ 59 ]
developed an assay for oligonucleotides where carboxyl-modified polystyrene
nanoparticles ( Ø 107 nm) were used as labels. The nanoparticle approach demon-
strated a 100-1,000-fold improvement in the detection limit compared to the
reference assay where biomolecules were labeled directly with the europium(III)
chelates. The same group reported a competitive immunoassay for estradiol [ 60 ]
which made use of polymeric beads stained with a luminescent europium(III)
complex. The beads were used as labels for the antibodies which interacted with
estradiol/Alexa Fluor 680 conjugates. Thus, FRET from the europium(III) complex
to Alexa Fluor 680 was inhibited in the presence of estradiol. Performance of the
immunoassays can be further improved by utilizing smaller beads based on the
copolymer of polystyrene and acrylic acid ( Ø 50 nm) [ 61 ]. As was demonstrated
recently, the smaller beads are much more efficient in FRET applications since the
amount of europuium complex located far from the surface and not participating in
FRET is significantly reduced [ 62 ]. Another group developed a fluorescent immu-
noassay for atrazine based on polystyrene beads stained with a europium(III)
chelate [ 63 ]. Europium(III) complexes encapsulated into Ormosil beads were also
shown to be promising for application in immunoassays [ 64 ]. Huhtinen et al. [ 65 ]
have demonstrated that luminescent complexes of other lanthanides (samarium,
terbium, dysprosium) can be used for staining polystyrene beads. Line type emis-
sion of the lanthanides can easily be separated which enables simultaneous deter-
mination of several analytes in immunoassays.
It should be mentioned that most of lanthanide chelates reported so far are
excitable in the UV region. Although autofluorescence is eliminated in time-
resolved measurements, high light scattering and absorption of light by biological
substances can be sometimes inconvenient. Therefore, development of visible
light-excitable probes is of much interest. Recently Wu et al. [ 66 , 67 ] encapsulated

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