Dye-concentrated organically modified silica nanoparticles as a ratiometric fluorescent pH probe by one- and two-photon excitation

Article abstract of DOI:10.1039/b600926c

Basic dye-concentrated nanoparticles (～ 33 nm in diameter) show fluorescence-based ratiometric pH response, by one- and two-photon excitations, with improved proton sensing ability (pK_a ～ 6.4) through nanoscopic intraparticle energy transfer. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b600926c

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COMMUNICATION
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Dye-concentrated organically modified silica nanoparticles as a
ratiometric fluorescent pH probe by one- and two-photon excitation{
Sehoon Kim, Haridas E. Pudavar and Paras N. Prasad*
Received (in Cambridge, UK) 23rd January 2006, Accepted 23rd March 2006
First published as an Advance Article on the web 6th April 2006
DOI: 10.1039/b600926c
would be densely dispersed everywhere through the nanoparticle,
where the proton-sensing event will take place mainly near the
surface. Given this localized event, the high-energy neutral and the
low-energy protonated states will closely coexist in the inner and
the surface parts of the nanoparticle, respectively, enabling the
inner-to-surface energy transfer. By virtue of such construction in
nano-space, the proton sensitivity, in terms of fluorescence, would
be improved by the energy transfer which amplifies the energy-
accepting protonated surface signal, and thus the apparent pKa
value of the nanoparticle will be modified from that of the dye
component. Pyridine-based dyes have potential for large two-
photon activity.8 Nevertheless, their use in pH probing, in general,
is restricted to rather acidic environments, as they are less sensitive
to protons, due to low pKa values.2,7 Thus, our strategy offers an
easy way to increase their low pKa values to a physiological level.
We report the preparation of NVP-concentrated ORMOSIL
nanoparticles and their intraparticle energy transfer characteristics.
Also demonstrated is the ratiometric pH-sensing behavior in the
near-physiological range, based on fluorescence emission by one-
and two-photon excitations.
Basic dye-concentrated nanoparticles (y 33 nm in diameter)
show fluorescence-based ratiometric pH response, by one- and
two-photon excitations, with improved proton sensing ability
(pKa y 6.4) through nanoscopic intraparticle energy transfer.
Growing demands on pH monitoring for environmental, biome-
dical, and bioprocess applications have driven the development of
fluorescence-based techniques in recent years, due to many
advantages which include greater sensitivity and ease of miniaturi-
zation, etc. This technique also benefits from the availability of
various natural and synthetic fluorescent probe molecules.1,2 In
particular, fluorescent indicators that exhibit pH-driven wave-
length alterations in their emission spectra allow for a more reliable
detection in a dual-emission ratiometric manner, which eliminates
data distortions caused by instrumental instability, photobleach-
ing, and probe concentration.2,3 However, most current pH
indicators display response only in fluorescent intensity, with the
exception of a few available probes including HPTS (8-hydroxy-
pyrene-1,3,6-trisulfonic acid), SNAFL (seminaphthofluoresceins)
and SNARF (seminaphthorhodafluors).1–3 Consequently, the
development of tailor-made ratiometric probes, by matching the
pKa to the pH of the experimental system, is gaining great interest
for better performances in various pH ranges of interest, for
example in intracellular monitoring, pH 6.8–7.4 in the cytosol and
4.5–6.0 in the cell’s acidic organelles.2,4 In cellular research,
nanosensors based on PEBBLEs (probes encapsulated by
biologically localized embedding) have been investigated due to
many advantages over free dyes, such as biocompatibility and
nontoxicity, etc.5
Synthetic procedures for NVP and its ORMOSIL nanoparticle
(OSNP) are depicted in Scheme 1. NVP was obtained exclusively
in a trans-form by Heck coupling between bromonaphthalene (1)
and 4-vinylpyridine. To achieve a large loading amount without
leak-out, NVP was functionalized into a silicate precursor
(Si-NVP) to be attached chemically to OSNP, by using 2-fold
In this paper, we propose the novel concept of a nanoparticle-
based fluorescent pH indicator for near-physiological use. The
designed particulate indicator is characterized by nano-assembly of
a pH-probing hydrophobic molecule, i.e., organically modified
silica (ORMOSIL) nanoparticle which is concentrated with a
ratiometric pH-responsive organic dye. ORMOSIL nanoparticles
are known as a water-dispersible and biocompatible carrier for
hydrophobic dyes in aqueous media.6 As for the hydrophobic dye,
we chose a naphthalenylvinylpyridine derivative (NVP, see
Scheme 1), where the pyridyl unit is a proton receptor. Like other
pyridine-based dyes,7 NVP responds to proton, with fluorescence
red-shift from high-energy blue to low-energy yellow. In the NVP-
concentrated ORMOSIL nanoparticle, it is expected that the dyes
Institute for Lasers, Photonics and Biophotonics, Department of
Chemistry, State University of New York, Buffalo, New York, 14260-
3000, USA. E-mail: pnprasad@buffalo.edu
{ Electronic supplementary information (ESI) available: Experimental
details, one-photon absorption, fluorescence, and excitation spectra of
NVP monomer and OSNP. See DOI: 10.1039/b600926c
Scheme 1 Reagents and conditions: i) HO(CH2)6Cl, K2CO3, KI, 18-
crown-6, DMF, 30 uC. ii) Pd(OAc)2, P(o-tolyl)3, Et3N, THF, 80 uC. iii)
2-fold ICTES, Et3N, dibutyltin diacetate, THF, reflux. iv) Hydrolysis,
NH4OH, THF, room temperature. v) Assembly into nanoparticle and sol-
gel condensation in aqueous micelle of AOT/1-butanol, room temperature.
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equimolar (3-isocyanatopropyl)triethoxysilane (ICTES). After
complete reaction, as determined by thin layer chromatography,
the resulting mixture was used for the next steps without
purification. The remaining excess ICTES acts as a co-monomer
for sol-gel polymerization, which possibly hinders close packing of
the NVP chromophores in the concentrated nanoparticle, to
minimize the intermolecular fluorescence quenching effect. OSNP
was obtained by sol-gel condensation in an aqueous micellar
medium of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and
1-butanol. After removal of surfactants by dialysis against water,
stable aqueous dispersions of OSNP were obtained with the help
of hydrophilic surface silanol groups.
depressed by basification (Fig. S1 in ESI{). Note that the actual
pKa value of NVP is not obtainable, owing to its water insolubility.
However, the above optical changes indicate that the population of
the protonated state under neutral conditions is so low as to be
slightly detected only by its three-fold more efficient emission,
suggesting its low pKa value.
It is also noteworthy that in Fig. 1a the red-shifted absorption
by protonation partially overlaps with the shorter-wavelength side
of the neutral blue emission. One can anticipate Fo¨rster-type
energy transfer from the neutral to the protonated NVPs, if the
two states exist in proximity, as in the case of OSNP. In solution,
however, such intermolecular interaction was not observed, when
examined with the partially protonated solution, by a certain
amount of citric acid, containing both neutral and protonated
NVP (Fig. S2 in ESI{). Since they are separated with each other by
solvation, the neutral and the protonated NVP contribute
independently to dual emission at 460 and 565 nm, respectively,
in a typical ratiometric manner. This independent behavior in
solution is no more the case for the NVP-concentrated OSNP,
where the emission spectrum is changed by nanoscopic intrapar-
ticle energy transfer (vide infra).
One- and two-photon optical properties of the NVP monomer
were examined in aqueous solution (Fig. 1a–b). A mixed solvent,
DMSO/water (5 1/1 by volume), was used to ensure homogeneous
dissolution in the micro-molar concentration range since the NVP
monomer is hydrophobic and not soluble in water. Fig. 1a shows
the optical responses of the NVP monomer to a change from
neutral to acidic or basic conditions. Upon acidification, absorp-
tion and fluorescence spectra are shifted to a longer wavelength by
protonation, due to the increased intramolecular charge transfer
(ICT) character. The fluorescence quantum yields (Wf) in the
neutral blue and the protonated yellow states were estimated as
0.06 and 0.18, respectively. Upon basification, absorption change
is negligible, while fluorescence exhibits a slight decrease in
intensity at the longer wavelength tail. The excitation spectra for
the tail emission at 600 nm, near the peak of the protonated
emission, show a noticeable change, where the protonated band at
ca. 400 nm was observed even in neutral conditions but was
The TPA cross-sections (s2) of the NVP solution in neutral and
acidic conditions were measured by the two-photon-induced
fluorescence method,9 using a femtosecond Ti-sapphire laser
(Fig. 1b). In the spectral window of 750–920 nm, the maximum
two-photon absorptivity in the neutral and the acidic conditions is
4 GM at 750 nm and 52 GM at 800 nm, respectively, where the
large enhancement for the latter is due to the increase in ICT
character. These values are comparable with those of commonly
used fluorescent probes such as fluoresceins and rhodamines,9
enabling two-photon application of the NVP-concentrated OSNP.
The spherically shaped OSNP with a diameter of 33 ¡ 6 nm
was successfully obtained in the micellar medium, as shown by the
transmission electron microscopic (TEM) image in Fig. 1c. In
neutral water, the OSNP dispersion exhibits the neutral NVP-like
absorption at 332 nm, with the baseline offset by Mie scattering,
while its fluorescence is positioned at 513 nm, between those of the
neutral and the protonated NVP monomers (Fig. 1d and Fig. S3
in ESI{). These optical properties of OSNP were maintained even
after 6 months of storage in the dark and were observed to be
stable during the measurements without any sign of photo-
degradation. Moreover, they are maintained even in an NVP-
solubilizing mixed solvent, DMSO/water (5 1/1 by volume),
supporting the formation of a rigid chemical construction within
the nanostructure (Fig. S3 in ESI{). The similarity of fluorescence
quantum yields between OSNP and NVP in the neutral mixed
solvent (Wf 5 0.04 and 0.06, respectively) implies that the excess
co-monomer, ICTES, is effectively minimizing the intermolecular
quenching process in the concentrated nanoparticle.
The unusually symmetric OSNP emission spectrum at pH 7
seems to be a mixed one, and thus can be analyzed into two peaks
derived from the neutral and the protonated contributions. This is
because, as mentioned above in relation to Fig. 1a, a certain
amount of the protonated NVP exists already even in neutral
conditions, undoubtedly on the surface in the case of OSNP.
Fig. 1d shows the result of peak separation using the spectral shape
of the OSNP emission at pH 4 for the longer-wavelength
protonated component. The calculated shorter-wavelength neutral
component at 494 nm is red-shifted with respect to the neutral
Fig. 1 (a) Normalized one-photon absorption and photoluminescence
(PL, excited at 370 nm) spectra of NVP in neutral (solid line), basic
(dashed line), and acidic (circle) conditions. (b) Two-photon excitation
spectra of NVP in neutral (triangle) and acidic (circle) conditions.
1 GM 5 10250 cm4s(photon-molecule)21. (c) TEM image of the obtained
OSNP. (d) Peak separation of the OSNP emission at pH 7 (solid line) into
the neutral (circle) and the protonated (dashed line) components.
2072 | Chem. Commun., 2006, 2071–2073
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to the intraparticle energy transfer which combines donor–
acceptor emissions apparently into a single peak, as discussed
above. The two-photon excitation of OSNP at 780 nm also
produces a similar fluorescence spectrum and pH-dependence,
except that the peak position and the intensity are slightly different
at every measured pH, due to the different two-photon
absorptivity of the NVP component between the neutral and the
protonated states. Despite a lack of noticeable dual emission, the
spectral evolution of one- and two-photon fluorescence of OSNP
enables ratiometric probing in the range of pH 4–8 at room
temperature, as shown in Fig. 2d. The intensity ratio of emissions
at 580 and 460 nm varies with pH, by one order of magnitude
under one-photon excitation and 3.4 times under two-photon
excitation. Importantly, OSNP, which includes a less proton-
sensitive pyridine-based dye, shows a greater response in the weak
acidic range by virtue of the improved proton sensitivity. From the
fitting of the titration curves, the same pKa values (y 6.4) were
extracted by both one- and two-photon excitation, which are
similar to the values obtained by a carboxyfluorescein probe, often
used for near-physiological pH measurement.2
In summary, the basic dye-concentrated nanoparticle structure
has been shown to improve the proton sensing ability, in terms of
fluorescence, through nanoscopic intraparticle energy transfer
from the neutral inner part to the protonated surface. We
anticipate that our approach, based on the dye-concentrated
ORMOSIL nanoparticle, will provide a promising pathway to
near-physiological intracellular pH monitoring, due to its advan-
tages which include water dispersibility, biocompatibility, and
appropriate pKa, as well as two-photon activity. Further, the
surface silanol groups of ORMOSIL nanoparticles can be
modified for targeted delivery, without affecting the intrinsic
properties of sensor molecules.
Fig. 2 pH-dependent spectral evolution of the OSNP dispersion: one-
photon absorption (a) and PL (b, excited at 370 nm), and two-photon
excited PL (c, excited at 780 nm). (d) Ratiometric plots of fluorescence
intensities at 580 and 460 nm by one- (circle, excited at 370 nm) and two-
(triangle, excited at 780 nm) photon excitations. The solid lines are the
fitting results for experimental data by using Eqn. (1) in ESI{.
NVP monomer emission at 460 nm, suggesting the occurrence of
energy transfer within OSNP. It is noted that, due to a partial
overlap in Fig. 1a, only the shorter-wavelength side of the neutral
NVP emission is transferred to the protonated one and the longer-
wavelength energy is left, resulting in a red shift of the neutral
component. Moreover, by virtue of energy transfer, the emission
from the protonated NVP on the surface, in spite of there being a
minimal amount, exhibits amplified intensity, comparable to the
excess neutral component, validating our nanoparticle-based
design strategy for improving proton sensitivity. The similarity in
the excitation spectra below 400 nm for both emission components
(at 450 and 600 nm, respectively) is evidence of intraparticle energy
transfer, indicating protonated emission generated by neutral
excitation (Fig. S4 in ESI{). Overall, the NVP-concentrated OSNP
emits greenish mixed fluorescence even under neutral conditions,
between the neutral blue and the protonated yellow ones of the
NVP monomer.
This work was supported in part by a DURINT grant from the
Chemistry and Life Sciences Directorate of the Air Force Office of
Scientific Research and in part by the Post-doctoral Fellowship
Program of Korea Science & Engineering Foundation (KOSEF).
Notes and references
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The spectral evolution and the ratiometric behavior of OSNP
under one- and two-photon excitation are shown in Fig. 2. The
absorption and the emission spectra of OSNP show practically
instantaneous responses to the pH change. The pH-dependence of
the one-photon absorption in Fig. 2a displays a dual-absorption
behavior, typical of ratiometric probes, with an isosbestic point at
ca. 370 nm, indicating the ground-state conversion from neutral to
protonated species. Upon excitation at 370 nm, however, OSNP
emits no normal dual emission, but broad, single-peak fluorescence
at every experimental pH, which becomes narrower and red-
shifted with decreasing pH. This unusual behavior, different from
dual emission of the NVP monomer (Fig. S2 in ESI{), is attributed
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Chem. Commun., 2006, 2071–2073 | 2073