Journal of the American Chemical Society
COMMUNICATION
In summary, we have described the synthesis, properties, and
cellular application of KS2, a new fluorescent chemosensor for
K+ sensing and imaging. As a result of the inclusion of the strong
electron-withdrawing group TCF with the TAC ligand, the new
sensor can respond to K+ over a broad concentration range up to
1600 mM. This is the first highly selective intracellular K+ sensor
that is capable of being used over such a broad range for highly
concentrated K+ sensing. Moreover, confocal microscopy experi-
ments established that KS2 can be used for detecting K+ levels
within living cells. The application of KS2 combined with other
sensors for cellular metabolism investigations and disease/cancer
detection and diagnosis is underway.
’ ASSOCIATED CONTENT
S
Supporting Information.
Synthesis, characterization,
b
and experimental details. This material is available free of charge
’ AUTHOR INFORMATION
Corresponding Author
Figure 4. (aꢀg) Time-dependent fluorescence of U87MG cells stimu-
lated by nigericin and observed under confocal fluorescence microscope:
(a) t = 0 (before the addition of nigericin); (bꢀg) t = 1, 3, 5, 10, 15, and
25 min, respectively, after the addition of nigericin (20 μM final
concentration) into the 20 mM KCl-containing medium. (h) Average
fluorescence intensity ratios as measured by ImageJ. I0 is the average
fluorescence intensity from (a); I is the average fluorescence intensity at
other times. An enlarged version of this figure is shown in S-Figure 10.
Author Contributions
†These authors contributed equally.
’ ACKNOWLEDGMENT
Financial support was provided by the Microscale Life
Sciences Center, an NIH Center of Excellence in Genomic
Sciences at Arizona State University (Grant 5P50 HG002360;
D.R.M. is the director and principal investigator).
an obvious fluorescence increase was observed after the cells
were treated with 5 μM isoproterenol and 20 mM KCl in
medium for 40 min at 37 °C (S-Figure 9). Isoproterenol has
been reported to stimulate potassium ion influx by stimulating
cyclic adenosine monophosphate (cAMP) generation, which is
associated with a physiological change in K+ transport.18 The
average fluorescence intensity of cells after the treatment was
33% greater than that before the treatment, as quantified using
ImageJ software (public domain software developed at the
National Institutes of Health).19
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The sensor was further demonstrated for in situ observation of
dynamic potassium ion influx and then efflux through changes in
fluorescence stimulated by an ionophore nigericin (Figure 4).
Cells internalized with 4 μM KS2 at 37 °C for 10 min were
washed with 20 mM KCl-containing fresh medium. Nigericin
(20 μM at its final concentration) in 20 mM KCl-containing
medium was added to help K+ to cross cell membrane. Enhanced
fluorescence was immediately observed after the addition of
nigericin, indicating that potassium ion influx occurred. The
potassium influx peaked after 3 min with an average fluorescence
increase of 370%. After 3 min, potassium efflux was observed
through a decrease in the fluorescence. The efflux stabilized after
15 min, and the fluorescence intensity after the stabilization was
about 25% below that before the stimulation by nigericin. The
ionophore nigericin was used to stimulate K+ influx for yeast
Saccharomyces cerevisiae.20 It was also reported to be a K+ efflux
stimulator for human colorectal adenocarcinoma HT-29 cells.4
The observation of the K+ influx and then efflux of U87MG cells
using KS2 demonstrates its further utility in measuring the
kinetics of K+ transport.
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dx.doi.org/10.1021/ja207345s |J. Am. Chem. Soc. 2011, 133, 18530–18533