Communications
2
+
in free intracellular Ca ions, followed by a decrease as a
result of receptor desensitization, thus clearly proving that the
calcium-sensor-loaded microspheres responded rapidly and
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1
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2
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A technique has thus been developed in which not only is
the acid salt of Indo-1delivered into cells but it is done so in a
manner in which dilution and leakage do not occur. An
experiment to demonstrate these advantages was performed
in which ND7 cells were incubated with either the micro-
spheres 9 and/or the commercially available Indo-1AM ester.
Both sensors were clearly visible within the cell at t = 0,
although the conventional dye was diffuse. The conventional
sensor was lost gradually with time, and only the sensors
bound to the microspheres were visible after 18 h of analysis
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[
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3449.
6
(
see the Supporting Information). These results indicate that
the sensor-based microspheres avoid the problems conven-
tionally associated with cellular leakage and allow intra-
cellular changes to be followed for long-term studies without
having to repeatedly load the cells.
1
13] M. Beierlein, K. R. Gee, V. V. Martin, W. G. Regehr, J. Neuro-
physiol. 2004, 92, 591– 599.
[14] R. M. Sanchez-Martin, M. Muzerelle, N. Chitkul, S. E. How, S.
Importantly, these beads are nontoxic to cells (see the
Supporting Information), do not disrupt cell physiology, and
can be introduced with high efficiency with the “capture” of
the sensor beads by the cells that allow cell sorting based on
bead content and fluorescence. There is of course a number of
questions that relate to the uptake of the beads and their
location within the cell. Recent reports in the area of
Mittoo, M. Bradley, ChemBioChem 2005, 6, 1341 – 1345.
[
15] G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem. 1985, 260,
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3
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[
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[19] J. C. Watkins, D. E. Jane, Br. J. Pharmacol. 2006, 147, S100 –
[
20,21]
[22]
derivatized-nanotube uptake
and bead uptake suggest
S108.
[
20] D. Pantarotto, R. Singh, D. McCarthy, M. Erhardt, J. P. Briand,
that the process is both size and material dependant, whereas
cellular location and compartmentalization also depend on
the amount of time the particles have been within the cell.
However, since spectroscopic changes on the beads are
observed following cellular stimulation, the sensors are
clearly chemically/biologically accessible and respond to
M. Prato, K. Kostarelos, A. Bianco, Angew. Chem. 2004, 116,
5
354 – 5358; Angew. Chem. Int. Ed. 2004, 43, 5242 – 5246.
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[
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[22] J. Rejman, V. Oberle, I. S. Zuhorn, D. Hoeskstra, Biochem. J.
2004, 377, 159 – 169.
2
+
changes in the concentration of intracellular Ca ions, thus
showing that they are located in the cytosol and not trapped
and isolated by a vesicular membrane.
By using these beads, we have shown our ability to
2
+
measure changes in the concentration of intracellular Ca
ions in single living cells in real time. The fact that the
microspheres are stably retained for several days also allows
2
+
changes in the concentration of intracellular Ca ions to be
followed in a single cell for much longer periods of time than
using traditional sensors. These results open the door to a
range of possible applications, with the microspheres acting as
carriers of sensors for other metal ions or pH values and for a
range of different biological probes.
Received: March 30, 2006
Published online: July 17, 2006
Keywords: biosensors · fluorescent probes · in vitro sensing ·
.
microspheres
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474
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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