Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 217

Steinkamp [ 91 ] for the Luminex microspheres. Recently, Mayr et al. stained
magnetic microspheres with luminescent ruthenium(II) metal ligand complexes
[ 92 ] which were encoded by time resolved imaging in the microsecond domain.
It should be mentioned that semiconductor nanocrystalls (quantum dots, QDs)
represent a powerful alternative to organic dyes here. The QDs of many colors can
be excited with a single light source and additionally benefit from size-tuneable
emission and high photostability. Similarly to fluorescent dyes the hydrophobic
QDs can be physically entrapped in polymeric beads (e.g., polystyrene) during
polymerisation [ 93 ] or be incorporated by swelling [ 94 ] or using layer-by-layer
deposition [ 95 , 96 ]. The combination of several QDs can theoretically result in
thousands of different codes [ 94 ], however, for many applications a few dozens of
codes is usually more than adequate [ 84 ]. In practice, the large number of codes
claimed is difficult to achieve because of the spectral overlap of the channels and
the need of a certain spectral region for the detection of the fluorescent label.
The dominated read-out technique of suspension arrays is flow-cytometry
(Fig. 6a ). This technique was originally developed for cell counting and sorting
and is ideally suited for the analysis of beads arrays. Particles are hydrodynamically
focused from a sample into a narrow stream in the center of the column. The
flowing stream of particles passes the beam of two lasers and the scattering light or
fluorescent light is detected. One beam quantifies the reporter fluorescence intensity
and the other is used to identify the beads. The most well known Luminex XMap
instrument uses orange and red stained micro-beads and a green reporter dye
(R-phycoerythrin). Particle analysis rates up to 10,000 per second can be obtained
and the sampling time for a 100-plex array is theoretically only 2-5 s (200-500
replicas) [ 97 ]. Among other companies, Becton Dickinson offers various sets of
beads to perform parallel assays on nonspecialised flow-cytometers with a multi-
plex capability of up to 30 ( http://www.bdbiosciences.com ).
A new suspension array concept based on sedimentation and microscopic
imaging was introduced by Moser et al. [ 98 ]. Magnetic microbeads settle to the
bottom of a microplate well by magnetic forces and form randomly ordered arrays,
which are examined by fluorescence microscopy and automated imaging analysis.
Each bead carries specific capture molecules and can be identified by a defined
luminescent code.
The number of multiplexed particles-based assays reported is manifold and
summarized in several reviews [ 85 , 86 , 97 , 99 ]. Bead sets for various nucleic acid
or protein assays are commercially available that are fully optimized for clinical
diagnostics and research purposes.
5.4.2 Randomly-Ordered Bead Arrays
In this type of arrays, microbeads carrying different indicator or receptor molecules
are randomly loaded into the wells of chemically etched optical fiber or silicon
chips [ 89 , 90 , 100 ] (Fig. 6b ). The beads are encoded with single or multiple
luminescent indicators or labels embedded into the polymeric core and/or bound