Measurement of Local Sodium Ion Levels near Micelle Surfaces with Fluorescent Photoinduced-Electron-Transfer Sensors

Article abstract of DOI:10.1002/anie.201509096

The Na⁺ concentration near membranes controls our nerve signals aside from several other crucial bioprocesses. Fluorescent photoinduced electron transfer (PET) sensor molecules target Na⁺ ions in nanospaces near micellar membranes with excellent selectivity against H⁺. The Na⁺ concentration near anionic micelles was found to be higher than that in bulk water by factors of up to 160. Sensor molecules that are not held tightly to the micelle surface only detected a Na⁺ amplification factor of 8. These results were strengthened by the employment of control compounds whose PET processes are permanently "on" or "off". The local Na⁺ concentration near an anionic tetramethylammonium dodecyl sulfate (TMADS) micelle surface was determined with new fluorescent photoinduced electron transfer (PET) sensors. Electrostatic interactions with the negatively charged sulfonate groups of the surfactant induce an increase in the Na⁺ concentration compared with bulk water.

Full text of DOI:10.1002/anie.201509096

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International Edition: DOI: 10.1002/anie.201509096
German Edition: DOI: 10.1002/ange.201509096
Micelles
Hot Paper
Measurement of Local Sodium Ion Levels near Micelle Surfaces with
Fluorescent Photoinduced-Electron-Transfer Sensors
Seiichi Uchiyama,* Eiko Fukatsu, Gareth D. McClean, and A. Prasanna de Silva*
Abstract: The Na+ concentration near membranes controls
our nerve signals aside from several other crucial bioprocesses.
Fluorescent photoinduced electron transfer (PET) sensor
molecules target Na+ ions in nanospaces near micellar
membranes with excellent selectivity against H+. The Na+
concentration near anionic micelles was found to be higher
than that in bulk water by factors of up to 160. Sensor
molecules that are not held tightly to the micelle surface only
detected a Na+ amplification factor of 8. These results were
strengthened by the employment of control compounds whose
PET processes are permanently “on” or “off”.
spacer–receptor” concept.[12] The benzo-15-crown-5 structure
was chosen as a receptor owing to its well-known binding
affinity towards Na+ (the logbNaþ value of benzo-15-crown-5
is 0.4 in water).[13] Importantly, the Na+ binding ability of the
receptor is not affected by changes in the environmental pH
value because it does not contain a pH-sensitive structure
(e.g., an amino group). Anthracene was used as a fluorophore
in 1a–1d owing to its high hydrophobicity, which facilitates its
uptake by micelles.[14] Furthermore, the anthracene structure
B
iological membranes organize living matter in cells, create
a fluid three-dimensional matrix, and allow for the controlled
transport of solutes.[1] Many biologically important processes
are membrane-mediated, yet surprisingly little is known
about both reaction schemes and, more fundamentally, the
complex nano-environments where reactions occur. For
instance, the gradient of Na+ concentration across biological
membranes is involved in the transport of molecules into cells,
pH homeostasis, and signal transmission in nerve systems,[2]
and is regulated by proteins such as Na+/K+ ATPases,[3] Na+/
H+ antiporters,[4] and voltage-gated Na+ channels.[5] Accurate
measurement of local Na+ levels near model membranes, such
as aqueous micelle surfaces,[6] requires sensors with excellent
selectivity against ubiquitous H+, but only very few are
available.[7] Although the concentration of membrane-
bounded H+ has been determined with fluorescent sensors,[8]
the concentrations of only a few larger ions (but not Na+)
have been measured near micelle surfaces and even then only
with sensors whose fluorescence output is influenced by H+.[9]
The related field of ion-driven, micelle-bound logic systems
has very few examples.[10] We have now determined local Na+
levels near a micelle surface with designed photoinduced
electron transfer (PET) sensors[11] for the first time.
The chemical structures of the fluorescent PET sensors
(1a–1d and 4a–4b) used in this study are shown in Figure 1.
These sensors were designed based on the “fluorophore–
[*] Dr. S. Uchiyama, Dr. G. D. McClean, Prof. A. P. de Silva
School of Chemistry and Chemical Engineering
Queen’s University
Belfast BT9 5AG (Northern Ireland)
E-mail: a.desilva@qub.ac.uk
Dr. S. Uchiyama, E. Fukatsu
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo Bunkyo-ku Tokyo 113-0033 (Japan)
E-mail: seiichi@mol.f.u-tokyo.ac.jp
Figure 1. Chemical structures of the fluorescent Na+ sensors (1a–1d
and 4a, 4b) and control compounds (2a–2d, 3, 5a, 5b, and 6) used
in this study. Counterions: BrÀ for 1a–1c and 2a–2c, ClÀ for 3.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201509096.
768
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participates in a non-radiative PET process when R1 is an
in their solubilities compared to those in water. The hypso-
electron-withdrawing substituent and the benzo-15-crown-5
unit is Na+ free.[15] Otherwise, it emits strong fluorescence.
Benzofurazan was also used as a fluorophore in some of the
sensors (4a, 4b). Along with the above characteristics
described for anthracene, the benzofurazan structure has
the remarkable property that the maximum emission wave-
length is dramatically shifted to shorter values in a hydro-
phobic environment.[8c,16] In both cases (1a–1d and 4a, 4b),
a short methylene spacer was used for efficient fluorescence
switching based on the PET mechanism, and the anchor
substituent was varied to change the local position of the
sensor within the membrane-bound nanoenvironment.
chromic shifts of the maximum emission wavelengths of the
benzofurazan compounds (4a, 4b, 5a, and 5b) in micellar
solution (Table 1) also indicate that these sensors and control
Table 1: Maximum emission wavelengths of 4a, 4b, 5a, and 5b.
Wavelength[a] [nm]
Solution
4a
4b
5a
5b
TMADS (20 mm)
CTAC (5 mm)
Triton X-100 (0.52 mm)
OG (34 mm)
573
573
546
575
594
573
574
541
563
n.d.[b]
573
573
563
573
595
572
573
558
564
n.d.[b]
water
2a–2d and 5a–5b are critically important control com-
pounds in studies of nanoenvironments by PET sensors as the
dimethoxybenzene moiety is unable to bind Na+ (i.e., the
fluorescence is always “off”). Thus the fluorescence proper-
ties of 2a–2d and 5a–5b can only be altered by salt-induced
environmental changes of the micelles (e.g., polarity
changes). 3 and 6 are additional control compounds that
never undergo PET (i.e., the fluorescence is always “on”).[17]
In the present study, a variety of anionic, cationic, and
neutral micelles were investigated as they all introduce
different nanoenvironments. The chemical structures of the
surfactants used in this study are shown in Figure 2. All of the
[a] Excited at the maximum absorption wavelength at 258C. [b] Could not
be determined because of low solubility.
compounds are in a hydrophobic environment, that is, close to
or inside the micelles. Representative fluorescence spectra of
the sensors and control compounds in micellar solutions are
shown in Figure 3 and 4. The most important result is that the
Figure 2. Chemical structures of the surfactants used in this study.
Critical micelle concentration (cmc): 5.5 mm for tetramethylammo-
nium dodecyl sulfate (TMADS),[22] 1.4 mm for cetyltrimethylammonium
chloride (CTAC),[23] 0.24 mm for Triton X-100,[23] and 25 mm for octyl b-
d-glucopyranoside (OG).[23]
Figure 3. Representative fluorescence spectra with a variation in Na+
concentration (pNa). a) 1a, b) 2a, c) 4a, and d) 5a (10 mm each) in
TMADS solution (20 mm). pNa refers to the total concentration in the
micellar solution and was varied by adding NaCl. The excitation
wavelengths (lex) are indicated in each panel.
micelles possess regions that are less polar than the surround-
ing aqueous environment but the presence of negatively
charged, positively charged, and neutral head groups has
a great influence over how the micelles interact with the
surrounding environment. For instance, Na+ ions are
expected to be localized near the negatively charged head
groups of the micelles because of electrostatic attraction.
The Na+ concentration near micelle surfaces was eval-
uated by studying the fluorescence properties of the sensors
as a function of bulk Na+ concentration. This method, which
had previously been applied to H+,[8] was used for Na+ sensing
for the first time. The interaction between the fluorescent
sensors (or control compounds) and the surfactants in
micellar solutions could be confirmed by a dramatic increase
fluorescence of the sensors is only switched on with increasing
Na+ concentration in TMADS solution (Figure 4; see also
Figure S1 and Table 2). When the control compounds are
used instead, such a behavior is not observed, indicating that
the fluorescence of the sensors is switched on by Na+ binding
rather by a change in the micellar nanoenvironment (e.g., the
local polarity) caused by salt effects.[18] However, the fluo-
rescence of the sensors is not switched by a change in Na+
concentration when they are placed in CTAC, Triton X-100,
or OG solutions. Although it was reported that the binding
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ogous to the DpKa method previously applied to micelle-
bound H+.[8a,20] The logbNaþ values of the sensors in micellar
solution were obtained using the equation:
log½ðImaxÀIÞ=ðIÀIminފ ¼ pNaÀlog bNaþ
where I, Imax, and Imin are the observed fluorescence intensity
at a fixed wavelength and the corresponding maximum and
minimum wavelengths, respectively (Table 2). Given the
modular behavior of fluorescent PET sensors,[12] the logbNaþ
value in water is 0.4 (the value for benzo-15-crown-5).[13] On
that basis, the DlogbNaþ values were calculated to be 2.0 (for
1a and 1b), 2.1 (1c), 0.9 (1d), and 2.2 (4a and 4b). Therefore,
the local Na+ concentration determined by the sensors near
the TMADS micelle surface is 7.9–158 (= 100.9–102.2) times
higher than that in bulk water. The range of micelle-bound
Na+ concentrations found by these sensors can be ascribed to
their different locations in the micellar system.[8c] Because of
the Na+ gradient created by the TMADS micelles, the neutral
sensor 1d would be located closer to the bulk water whereas
the other cationic sensors are found closer to the micelle
surface owing to ion paring with the head group. If the
position of the sensors could be adjusted more extensively by
structural variants of 1a–1d and 4a–4b,[8c] our method would
allow for the determination of the Na+ gradient near the
micelle in more detail. The development of new receptors
with stronger binding affinities towards Na+ in aqueous media
is also important because fluorescent sensors with such
receptors will be able to determine the Na+ concentration
even near neutral and cationic micelles where Na+ is repelled
in comparison to bulk water because of dielectric effects and/
or an electrostatic repulsion.
Figure 4. Dependence of the fluorescence quantum yield (Ff) on pNa
*
for a) 1a, b) 2a, c) 4a, and d) 5a (10 mm) in TMADS (20 mm, ),
~
*
CTAC (5 mm, ), Triton X-100 (0.52 mm, ), and OG (34 mm, ”)
solutions. pNa refers to the total concentration in the micellar solution
and was varied by adding NaCl.
Table 2: Fluorescence properties of the sensors and control compounds
in TMADS solution.
[a]
[b]
Compound
Ff, high ½Naþ
Ff, low ½Naþ
FE[c]
logbNaþ
Š
Š
anthracenes
1a
2a
1b
2b
1c
2c
1d
2d
3
0.54
0.32
0.66
0.17
0.81
0.43
0.045
0.010
0.81
0.27
0.29
0.24
0.17
0.34
0.35
0.0056
0.0092
0.78
2.0
2.7
2.4
8.1
2.41Æ0.18
2.35Æ0.02
2.50Æ0.04
1.26Æ0.15
In conclusion, a series of new fluorescent PET sensors
have been used to measure local Na+ concentrations that are
electrostatically increased in nanospaces[21] near anionic
micelles for the first time. Similar experiments in nanospaces
near more biorelevant membranes such as vesicles and
liposomes are currently being conducted in our laboratories.
Another important step for future biological use would be to
improve the Na+/K+ selectivity of these sensors while
preserving the pH independence. The diversity of available
fluorescent PET sensor components for various targets[11,12]
will also allow us to measure the concentrations of other
important ions in biological nanoenvironments in a similar
manner.
benzofurazans
4a
5a
4b
5b
6
0.045
0.025
0.057
0.031
0.040
0.028
0.029
0.038
0.039
0.047
1.6
1.5
2.57Æ0.02
2.56Æ0.03
[a] Na+ concentration in solution: 0.1m. [b] Na+ concentration in
solution: 0m. [c] Fluorescence enhancement factor: Ff, high ½Naþ
/
Š
Ff, low ½Naþ
.
Š
ability of benzo-15-crown-5 towards Na+ increased in less
polar media,[13] this effect was not observed even in the Triton
X-100 micelles, which create the most hydrophobic environ-
ment for the sensors in the present study (see the maximum
emission wavelengths in Table 1). This observation is due to
the fact that ions avoid less polar membranes, preferring
adjacent aqueous regions instead.[8c] Therefore, we conclude
that Na+ ions are mainly localized near TMADS micelles
because of electrostatic attraction to the negatively charged
sulfonate groups of the surfactant.
Acknowledgements
We thank the Japan Society for the Promotion of Science for
Grants-in-Aid for JSPS Fellows (02J02483) and Young
Scientists (A) (25708023) as well as the Department of
Employment and Learning, Northern Ireland.
Keywords: fluorescence spectroscopy · fluorescent probes ·
micelles · sensors · sodium
The local Na+ concentration near TMADS micelles was
measured by determining the D(logbNaþ ) value (logbNaþ in
TMADS solutionÀlogbNaþ in water) of the sensors,[19] anal-
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Received: September 28, 2015
Published online: October 27, 2015
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