Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 211

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric
beads to be used as an alternative for ion sensing. For example, a reporter protein
(composed of a phosphate-binding protein, a FRET donor (cyan fluorescent pro-
tein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into
polyacrylamide nanobeads by Sun et al. [ 46 ]. FRET was inhibited upon binding
of phosphate. Kopelman and co-workers [ 47 ] used a similar approach to design a
nanosensor for copper ions. They have found that fluorescence of red fluorescent
protein DsRed (commonly used as a label) is reversibly quenched by Cu 2+ and Cu + .
Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into
polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably
measured. It should be mentioned, that in contrast to much more robust dyes, mild
conditions upon polymerization and purification are very important for immobili-
zation of the biomolecule to avoid degradation.
Gouanve et al. [ 9 ] presented another approach to designing copper nanosensors.
They prepared cross-linked polystryrene beads ( Ø 14 nm) and functionalized the
surface with 1,4,8,11-tetraazacyclotetradecane (Cyclam), which selectively bound
copper ions. The core of the beads was stained with a lipophilic fluorescent dye
9,10-diphenylanthracene by swelling. Fluorescence of the dye was quenched in the
presence of Cu 2+ due to FRET. The particles were suitable for sensing Cu 2+ in

micromolar concentrations.

5.1.4 Beads for Sensing and Imaging of Metabolites

Kopelman and co-workers [ 48 ] attempted to design a nanobiosensor for glucose by
immobilizing an oxygen indicator, ruthenium(II) tris(4,7-diphenyl-1,10-phenan-
throline disulfonic acid) dichloride, glucose oxidase and a reference dye into
polyacrylamide beads ( Ø 45 nm). An increase of luminescence intensity of the
oxygen indicator was observed in the presence of glucose due to the consumption of
oxygen in the enzymatic reaction. The calibration plot remained linear between 0.3
and 5 mM of glucose. However, this is at variance with the results of Stein et al. [ 49 ]
who found that even in case of much larger silica/alginate microbeads ( Ø 14

m)
doped with an oxygen indicator (PtOEP) and glucose oxidase the diffusion of
glucose is too fast for the sensor to operate in the physiologically relevant dynamic
range (2-20 mM of glucose). The beads were found to respond linearly only from
0.1 to 5.5 mM of glucose. Additional polyelectrolyte bilayers poly(allylamine
hydrochloride)/poly-(sodium 4-styrenesulfonate) were necessary to control the dif-
fusion of the analyte and the linear dynamic range was extended to 11 mM of
glucose [ 50 ]. Moreover, a rhodamine derivative was entrapped into the polyelec-
trolyte nanolayers to enable ratiometric referencing.
Zenkl et al. [ 51 ] presented another approach to design saccharide-sensitive nano-
beads. They prepared particles ( Ø 380 nm) based on poly( N -isopropylacrylamide)
cross-linked with phenylboronic acid moieties. In the presence of a saccharide
(glucose or fructose) the particles reversibly swell due to the formation of negative
charges. A FRET-indicator couple (fluorescein/rhodamine) is used to monitor the

m