A ratiometric probe for the selective time-gated luminescence detection of potassium in water

Article abstract of DOI:10.1039/c0cc02637a

A europium probe for the ratiometric detection of potassium in water is presented. This probe demonstrates high sensitivity, with an affinity for K ⁺ in the mM range, and high selectivity for K⁺ over Na⁺, Ca²⁺, Mg²⁺ and Li⁺. The long luminescence lifetime of the probe and its large Stokes shift further enable accurate determination of the concentration of K⁺ in complex aqueous media.

Full text of DOI:10.1039/c0cc02637a

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A ratiometric probe for the selective time-gated luminescence detection
of potassium in waterwz
´
Evan A. Weitz and Valerie C. Pierre*
Received 18th July 2010, Accepted 9th November 2010
DOI: 10.1039/c0cc02637a
A europium probe for the ratiometric detection of potassium in
water is presented. This probe demonstrates high sensitivity,
with an affinity for K+ in the mM range, and high selectivity for
K+ over Na+, Ca2+, Mg2+ and Li+. The long luminescence
lifetime of the probe and its large Stokes shift further enable
accurate determination of the concentration of K+ in complex
aqueous media.
develop probes that could potentially be used to image the
action potentials and other processes which involve rapid
fluctuation in K+ levels. In this regard, our group recently
developed a highly selective luminescent probe based on the
cation–p interaction and the dependence of the terbium
luminescence on the distance separating the metal from its
antenna.6 Unfortunately, the effectiveness of this probe in
aqueous solutions was limited. Based on the work of the
Parker group, we reasoned that a selective turn-on probe
could be developed via direct coordination of the potassium
ion by a phenanthridine antenna. Parker and coworkers
previously reported that the excitation profile of a macrocyclic
EuIII complex bearing a phenanthridine antenna varied
significantly with the latter’s protonation state.7 Protonation
of the phenanthridine resulted in a complex that could be
excited at significantly longer wavelength. Moreover, the
presence on an isosbestic point in the excitation profiles
suggested that a phenanthridine antenna would lead to a
ratiometric probe. A ratiometric probe would be significantly
more valuable in the accurate measurement of potassium
concentration in living cells since the concentration of such a
probe in biological studies can be readily determined.
The development of luminescent probes for the imaging of
potassium inside and outside living cells remains an active and
challenging area of research. Given the importance of the
alkali ions in a multitude of biological processes, including
regulation of membrane polarization and osmotic pressure,
there is a need for practical sensitive and selective luminescent
probes for K+. However, sensitivity and selectivity alone are
not sufficient to ensure that a probe be valuable for neuroimaging.
The probe must also turn on and off with speed comparable to
the flux of the alkali ions across the cell membrane. This
requires that the kinetics of binding and dissociation of the
K+-probe adduct also be comparable to the rate of flow of the
ions during the propagation of an action potential. Such
probes, presenting high sensitivity and selectivity with fast
binding and dissociation kinetics, have yet to be developed.
Herein, we present a step towards this goal with the
development of a ratiometric probe for K+ that displays high
selectivity and sensitivity in the mM range by incorporating a
phenanthridine-based lariat ether.
Our probe, Eu-KPhen (1) (Fig. 1) thus incorporates a
macrocyclic Eu complex for improved stability in biological
media, a diaza-18-crown-6 receptor, and a phenanthridine
antenna conjugated to the crown ether at the 4 position.
Conjugation at this position enables the phenanthridine to
coordinate the potassium encapsulated within the crown ether.
We hypothesized that this complexation would modify the
excited energy levels of the antenna, thus affecting the
excitation profile of the Eu complex resulting in a ratiometric
turn-on probe (Fig. 1).
The majority of luminescent probes for potassium
developed thus far rely on PeT (photo electron transfer)
quenching of a chromophore or an antenna by a nearby
amine-containing receptor.1–4 The selectivity of these probes
are thus entirely based on that of the receptor. They can be
divided into two classes: crown- or lariat-ethers, which
demonstrate limited selectivity for K+ over Na+ but rapid
turn-on kinetics; and cryptands which show improved
selectivity but also significantly slower binding and dissociation
rates.5 This slow cation release in turn prevents the application
of the cryptand-based probes for the imaging of very rapid
fluctuation in K+ concentrations, such as those that occur
during an action potential.
The probe Eu-KPhen (1) was synthesized in thirteen steps
according to Scheme 1 (see ESI for details).z The europium-
centered time-gated luminescence intensity of Eu-KPhen (1)
increases up to two fold upon titration of potassium ion in
water at neutral pH when excited at 267 nm (Fig. 2). Remarkably,
the binding affinity of Eu-KPhen (1) for K+ in water is
We aimed to improve the selectivity of lariat-ether based
receptors without affecting their kinetics of binding so as to
Department of Chemistry, University of Minnesota, 207 Pleasant
Street SE, Minneapolis, MN 55455, USA. E-mail: pierre@umn.edu;
Fax: (+1) 612 626 7541; Tel: (+1) 612 625 0921
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Detailed
experimental procedure for the synthesis of Eu-KPhen and titrations.
See DOI: 10.1039/c0cc02637a
Fig. 1 Chemical structure and mode of action of the K+ probe
Eu-KPhen (1).
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concentrations ranging from 0–10 mM in water at physio-
logical pH. As such, the probe is ideally suited for imaging the
alkali cation in the extracellular environment, where its
concentration ranges typically ranges from 3.5–5.3 mM.9 It
should be noted that for application in neuroimaging, it is
more beneficial to image the extracellular K+ level than
intracellular ones, as the bigger concentration change during
an action potential will occur extracellularly. In this respect,
Eu-KPhen (1) is a significant improvement over the previously
published lanthanide-based probes of Gunnlaugsson3
and Wong4 which enable detection of aqueous K+ in the
0.1–1 M range but which displayed negligible luminescence
enhancement at the mM ranges of extracellular significance.
Eu-KPhen (1) has three other significant advantages over
previously reported luminescent probes for potassium: (1) it is
ratiometric, (2) it has a long luminescence lifetime, and (3) it
displays a very large pseudo-Stokes shift between the absorption
of the antenna and the emission of the lanthanide. As can be
seen in the excitation profile of the europium complex as a
function of the concentration of added potassium (Fig. 3), if
the europium complex is excited at shorter wavelengths such
as 265 nm, then its emission depends on the concentration of
K+. Excitation at 400 nm, however, yields a europium
centered emission which is independent of the concentration
of potassium. The two excitation wavelengths can thus be used
ratiometrically to determine quantitatively and thus with
significantly higher accuracy, the concentration of potassium
in a biological medium. Excitation at 400 nm yields the
concentration of the europium probe. From the binding
affinity of Eu-KPhen for K+ (26 mMꢁ1), the emission intensity
of Eu upon excitation at 267 nm enables accurate determination
of the concentration of the alkali cation.
Scheme 1 Synthesis of Eu-KPhen (1). Reagents and conditions:
(a) PhB(OH)2, (PPh3)4Pd, Na2CO3, toluene, H2O, reflux, 13 h,
92.9%; (b) 36 bar H2, Pd/C, CH3OH, 21 h, 99.0%; (c) HCOOH,
N2, reflux, 52 h; (d) PPA, 1501 C, 3 h, 68.5% over 2 steps; (e) NBS, hn,
benzene, rt, 51.4%; (f) Cs2CO3, CH3CN, 501 C, 96 h, 44.8%; (g) TFA,
CH2Cl2, rt, 1.5 h, 83.2%; (h) chloroacetylchloride, N2, CH2Cl2, 01 C,
4 h, 63.5%; (i) cyclen, Cs2CO3, CH3CN, Ar, rt, 88 h; (j) t-BuO2CCH2Br,
CH3CN, rt, 59.5% over 2 steps; (k) TFA, CH2Cl2, rt, 1 h, 80.9%;
(l) EuCl3, H2O, pH 7, reflux, 19 h, 99.9%. See ESI for details.z
Comparison with the UV-visible absorption spectrum of the
phenanthridine moiety (see ESIz) indicates that the responsive
wavelengths of excitation of the probe Eu-KPhen (250–350 nm)
correspond to the absorbance of the phenanthridine moiety.
Coordination of K+ by the phenanthridine thus likely affects
the efficiency of energy transfer from the antenna to the
lanthanide. Correspondingly, the intensity of Eu emission is
a function of the K+ concentration, as observed in the
titrations (Fig. 2).
Additionally, the long luminescence lifetime of europium
(>1 ms) readily enables time-gated luminescence imaging,
Fig. 2 Relative time-delayed luminescence of Eu-KPhen (1) as a
function of K+ concentration (filled black squares) and Na+
(open red circles). Error bars represent standard deviation (s.d.),
n = 3. Experimental conditions: [Eu-KPhen] = 50.0 mM, 25 mM
aqueous OAcꢁ buffer pH 7, excitation at 267 nm, emission at 593 nm,
time delay = 0.1 ms, T = 20 1C.
26 ꢀ 0.8 mMꢁ1. This is notably higher than the affinity of the
corresponding diaza-18-crown-6 and lariat ethers for the alkali
cation which are typically in the mMꢁ1 range,8 and therefore
suggests that at least partial coordination of the potassium by
the phenanthridine’s nitrogen is occurring. Advantageously,
this higher binding affinity enables the detection of K+ in
Fig. 3 Metal luminescence excitation spectra of Eu-KPhen (1) upon
titration of K+. Experimental conditions: [Eu-KPhen] = 50.0 mM,
25 mM aqueous OAcꢁ buffer pH 7, emission 593 nm, time delay =
0.1 ms, T = 20 1C.
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a large excess of Li+ (50.0 mM) also has negligible influence
on the luminescence of the probe. Importantly, Importantly,
the subsequent addition of 20 mM KOAc restores the two-fold
increase in luminescence, demonstrating that the presence of
competing cations does not affect the determination of K+
concentration. (see ESIz).
In conclusion, we have developed a novel lanthanide-based
luminescent probe for the detection of potassium in water at
neutral pH. This probe presents high affinity for K+ in the
mM range and good selectivity over Na+ and physio-
logically relevant cations including Ca2+, Mg2+ and Li+.
Advantageously, this probe presents long luminescence life-
time which readily enables time-gating imaging. Notably, it is
also the first ratiometric luminescent probe for K+; the
Eu-centered luminescence is dependent on the K+ concentra-
tion if the probe is excited at 265 nm but independent of it if
excited at 400 nm. The ratiometric response enables a more
accurate determination of K+ levels in solutions.
Fig. 4 Selectivity of Eu-KPhen (1) for K+ over physiological cations
at typical serum concentrations. Time-gated emission spectra (1) in
water (black) and in the presence of 50.0 mM Li+ (blue), 0.83 mM
Mg2+ (green), 2.47 mM Ca2+ (yellow), 107 mM Na+ (orange) and
10 mM K+ (red). Experimental conditions: [Eu-KPhen] = 50.0 mM,
25 mM aqueous OAcꢁ buffer, pH 7, excitation at 267 nm, emission at
593 nm, time delay = 0.1 ms, T = 20 1C.
whereby any background luminescence from a biological
media, which typically has a lifetime shorter than the ms range,
is advantageously cut-off.10 This techniques further improves
the accuracy of the probe in biological applications.
Moreover, the large Stokes’ shift of the complex (excitation
at 267 nm, emission at 593 nm) and the negligible overlap
between the absorption and the emission spectra, limit any
self-absorption problems at high concentration. Taken
together, these advantages render Eu-KPhen (1) a promising
candidate for imaging extra-neuronal potassium fluxes during
an action potential.
This research was supported by the University of
Minnesota.
Notes and references
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For application in neuroimaging, however, a potassium
probe must also be highly selective for potassium over sodium.
The extracellular concentration of potassium is 3.5–5.3 mM
whereas that of sodium is 135–148 mM. There is therefore a
30–40 fold excess of sodium in the extracellular matrix. As can
be seen in Fig. 3, the selectivity of Eu-KPhen (1) for K+ over
Na+ is significantly higher than that of most crown and lariat
ethers, which typically demonstrate 5–10 fold selectivity for
the larger alkali cation.8 The time-gated luminescence intensity
of the probes increases 83% upon addition of K+ and
plateaus at 10 mM, whereas negligible turn on is observed
with a similar range of sodium concentrations (Fig. 3).
A typical extracellular concentration of 137 mM Na+, increases
the luminescence by 35%. In comparison, the luminescence
intensity of Tsien’s PBFI probe11 and Crossley’s CD18 probe1
in the presence of 10 mM K+ is similar to that of 125 mM Na+.
The higher selectivity for K+ vs. Na+ and the ratiometric
aspect of Eu-KPhen (1) renders it a more promising candidate
for the intended long-term application of imaging neuron
activity.
The selectivity of Eu-KPhen (1) for K+ over other
physiological cations including Ca2+, Mg2+ and Li+ is shown
in Fig. 4. Typical serum concentration of the alkali earth9
Ca2+ (2.47 mM) and Mg2+ (0.83 mM) do not significantly
affect the response of the probe. Ca2+ increases the luminescence
intensity by 9% whereas Mg2+ increases it by 3%. Addition of
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11 A. Minta and R. Y. Tsien, J. Biol. Chem., 1989, 264, 19449–19457.
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