Water-Soluble Osmium Complexes Suitable for use in Luminescence-Based, Hydrogel-Supported Sensors

Article abstract of DOI:10.1007/s10895-016-1923-z

Osmium transition metal complexes are of particular interest in luminescence-based sensing applications because of their longer wavelength absorptions and emissions, relative to similar ruthenium and rhenium complexes, that allow for inexpensive excitation and minimize interferences from autofluorescence when the sensor is used in biological samples. Reported here are the photophysical properties of a series of water-soluble osmium complexes suitable for use in hydrogel-based sensors: [Os(bpy)₂(sulf-dpp)]Cl₂, [Os(phen)₂(sulf-dpp)]Cl₂, [Os(dpp)₂(sulf-dpp)]Cl₂, and [Os(CO)₂Cl₂(sulf-dpp)], where bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline, dpp is 4,7-diphenyl-1,10-phenanthroline, and sulf-dpp is bathophenanthrolinedisulfonic acid disodium salt. The family of complexes showed minimal oxygen quenching, making them particularly well-suited for sensing applications in which oxygen concentration varies. Luminescence anisotropy was found to depend more significantly on net dipole moment than hydrodynamic radius of the molecule, and, as expected, excited state lifetime and luminescence anisotropy were highly dependent on the local environment of the reporter molecule. Results obtained for hydrogel-based relative humidity sensors containing [Os(CO)₂Cl₂(sulf-dpp)] and [Os(bpy)₂(sulf-dpp)]Cl₂ complexes highlight the significant potential for this class of compounds in a hydrogel-supported luminescence-based sensing approach.

Full text of DOI:10.1007/s10895-016-1923-z

J Fluoresc
DOI 10.1007/s10895-016-1923-z
ORIGINAL ARTICLE
Water-Soluble Osmium Complexes Suitable
for use in Luminescence-Based, Hydrogel-Supported Sensors
Amy M. Wagner1 & Sarah A. Strohecker1 & Elizabeth K. Costello1 & Jeffrey A. Rood1
&
Kristi A. Kneas1
Received: 27 June 2016 /Accepted: 26 August 2016
#
Springer Science+Business Media New York 2016
.
Abstract Osmium transition metal complexes are of particu-
lar interest in luminescence-based sensing applications be-
cause of their longer wavelength absorptions and emissions,
relative to similar ruthenium and rhenium complexes,
that allow for inexpensive excitation and minimize in-
terferences from autofluorescence when the sensor is
used in biological samples. Reported here are the
photophysical properties of a series of water-soluble os-
mium complexes suitable for use in hydrogel-based sen-
sors: [Os(bpy)2(sulf-dpp)]Cl2, [Os(phen)2(sulf-dpp)]Cl2,
[Os(dpp)2(sulf-dpp)]Cl2, and [Os(CO)2Cl2(sulf-dpp)], where
bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline, dpp is 4,7-
diphenyl-1,10-phenanthroline, and sulf-dpp is
bathophenanthrolinedisulfonic acid disodium salt. The
family of complexes showed minimal oxygen
quenching, making them particularly well-suited for
sensing applications in which oxygen concentration
varies. Luminescence anisotropy was found to depend
more significantly on net dipole moment than hydrody-
namic radius of the molecule, and, as expected, excited
state lifetime and luminescence anisotropy were highly depen-
dent on the local environment of the reporter molecule.
Results obtained for hydrogel-based relative humidity sensors
containing [Os(CO)2Cl2(sulf-dpp)] and [Os(bpy)2(sulf-
dpp)]Cl2 complexes highlight the significant potential for this
class of compounds in a hydrogel-supported luminescence-
based sensing approach.
Keywords Osmium transition metal complexes
.
.
Luminescence-based sensors Hydrogel Luminescence
anisotropy
Introduction
Because of their high selectivity and sensitivity,
luminescence-based sensors have proven useful for a wide
variety of applications including humidity sensing, detection
of nitro-based explosives, and detection of ATP in human
serum [1–3]. Analyte concentrations have been determined
from measured changes in emission intensity, emission wave-
length, excited state lifetime, and luminescence anisotropy
[4–10]. Despite an extensive body of literature devoted to
the development of luminescence-based sensors, there remain
some design challenges. Notable are the limited availability of
analyte-responsive luminophores and the relative difficulty of
engineering new ones [11, 12]. In addition, few sensing com-
pounds are suitable for use in aqueous solution or in the de-
tection of vapor, as required for biological and environmental
applications [13–15]. Given that many luminescence-based
sensors make use of a luminophore embedded within a poly-
mer matrix, a possible way to address the aforementioned
limitations is to shift the analyte sensitivity and selectivity to
the polymer matrix, over which there is great synthetic con-
trol, and use the luminophore as a secondary reporter. A gen-
eralized luminescence-based sensing strategy using an
environment-sensitive luminophore embedded within a hy-
drogel was reported previously to measure relative humidity
[1], and the approach could be modified to incorporate a smart
hydrogel which responds to analytes such as glucose, lactate,
and hydrogen [16–18]. In the approach, the hydrogel swells
and contracts with changing analyte concentration, thereby
changing the local environment of the luminophore (i.e.,
* Kristi A. Kneas
kneask@etown.edu
1
Department of Chemistry & Biochemistry, Elizabethtown College,
One Alpha Drive, Elizabethtown, PA 17022, USA

J Fluoresc
polarity and/or rigidity) and its emission properties, which can
be monitored to quantitate analyte.
[5, 20]. In addition they have more limited sensitivity to
changes in temperature or oxygen concentration [5, 23].
Osmium complexes similar to those reported here have
also proven useful in applications such as solar cells, light
emitting diodes, and immunoassays where the value of osmi-
um complexes has been highlighted [24–26]. The utility of
osmium complexes as biomolecular probes stems from their
long wavelength absorption and emission wavelengths, which
minimize interference from autofluorescence in biological
systems, and their long excited state lifetimes which are suit-
able for investigation of DNA dynamics, domain-to-domain
motions in proteins, rotational motion of lipid vesicles, and
antibody binding in immunoassays [4]. Applications such as
these also benefit from the ability to tune the photochemical
and photophysical properties of luminescent osmium com-
plexes. [27, 28] To improve probes for use in hydrogel-
based sensors, environment-sensitive TMCs must be prepared
with high anisotropies and hydrodynamic radii matching the
void volume in the hydrogel when swollen over the analyte
concentration range of interest. Ligand tuning can also
be used to control lipophilicity and hence the partitioning of
TMCs between aqueous phase and membranes [29], thus in-
dicating the possibility of positioning TMCs at the hydrogel-
aqueous solution interface to improve sensitivity in hydrogel-
based sensors.
To exploit the full potential of this generalized sensing
approach, the environment-sensitive luminophore must be op-
timized. Organic fluorophores like dapoxyl sulfonic acid
(DSA), which was used previously [1], are beneficial because
of high quantum yields but prove less optimal because of poor
photostability and short wavelengths of absorption and emis-
sion. The former limits the longevity of a sensor, and the latter
presents difficulty in biological applications where autofluo-
rescence is an issue [4]. Since there is no covalent attachment
of the fluorophore to the hydrogel network, an added chal-
lenge is the loss of fluorophore via leaching when the hydro-
gel swells and contracts in response to analyte. Because
intensity-based sensors are prone to errors resulting from
changing concentration of luminophore in the path of exciting
light, more desirable would be luminophores that exhibit ap-
preciable changes in emission wavelength, excited state life-
time, or luminescence anisotropy, with the last two having
potential for greatest sensitivity. Luminescence anisotropy (r,
Eq. 1) is determined from the ratio of emitted polarized light
relative to the total light emitted when the sample is excited
with polarized light. In Eq. 1, I⊥ is the intensity measured
perpendicular to the incident light, and I∥ is the intensity mea-
sured parallel to the incident light.
In the work reported here, the goal was to prepare and
characterize water-soluble osmium complexes with high fun-
damental anisotropy, long emission wavelengths, and long
excited state lifetimes for use in hydrogel-based sensors.
Given the nature of these osmium alpha diimine complexes,
a number of these design features are in competition with one
another. Figure 1 shows a simplified orbital diagram and typ-
ical triplet state orderings for osmium complexes containing
alpha-diimine ligands such as 2,2′-bipyridine, 1,10-
phenanthroline, and 4,7-diphenyl-1,10-phenanthroline. As is
the case for the widely utilized family of ruthenium (II)
polypyridal complexes, the lowest excited state in osmium
complexes of this type is typically a charge transfer state.
[20, 30, 31] There are, however, two important differences
in the orbital diagram for osmium as compared to that of
ruthenium. Crystal field splitting, which increases when de-
scending a group, is significantly greater for Os than for Ru,
thereby resulting in significantly higher d-d states for osmium
complexes. Resultant benefits are greater photostability and
minimal temperature-dependence since the d-d states are ther-
mally inaccessible from the emitting MLCT state. Easier ox-
idation of osmium relative to ruthenium also means that the
energy of the MLCT state is significantly lower than that of
analogous ruthenium complexes. A favorable consequence is
longer emission wavelengths, but a disadvantage is the in-
creased efficiency of quenching to the ground state and thus
reduced quantum yield and shorter excited state lifetimes of
these types of osmium complexes, in accordance with the
I∥−I⊥
I∥ þ 2I⊥
r ¼
ð1Þ
Dipole moment and hydrodynamic radius of a complex
influence anisotropy because of their respective impacts on
photoselective excitation by the polarized light and the extent
of depolarization of the emission by rotational diffusion.
Solvent viscosity also plays a role, with an increase in viscos-
ity resulting in an increased anisotropy because of reduced
rotational diffusion of the emitting compound [19].
As a category of luminescent reporter compounds, transi-
tion metal complexes (TMCs), most notably those of the plat-
inum metals of Ru, Os, Re, Rh, and Ir have been widely used
[20, 21] and meet the demands of the current application.
They are thermally and chemically stable, photochemically
robust, and have intense visible absorptions, relatively long
excited state lifetimes, large Stokes shifts, and reasonable
quantum yields, which afford easier measurement and im-
proved sensitivity [4, 20]. Also significant, the luminescence
properties of transition metal complexes, including their envi-
ronment-sensitivity, emission wavelength, excited state life-
time, and fundamental anisotropy, can be tuned by alteration
of the ligands [20, 22]. Osmium complexes are of particular
interest in the current work because they typically have longer
wavelength absorptions and emissions than other luminescent
TMCs, thus allowing inexpensive excitation and minimizing
interferences from autofluorescence in clinical samples

J Fluoresc
M(eg)
bathophenanthroline (98 + %), 1,10-phenanthroline (99 %), and
acrylamide (99.9 %) were purchased from Alfa Aesar.
Formaldehyde (37 % aqueous solution stabilized with 10–
15 % methanol), formic acid (98 %), N,N′-methylene-bis-acryl-
amide (98 + %), 2-(dimethylamino)ethyl methacrylate (98 %),
potassium persulfate (99 + %), and Sephadex G-50 were pur-
chased from Sigma-Aldrich. Ethanol (Certified ACS), acetone
(NF grade), and ethyl ether (Reagent grade ACS) were pur-
chased from Pharmco-Aaper, and methanol (Reagent grade
ACS) was obtained from Carolina Biological Supply
Company. GFS Chemicals was the source of 2,2′-bipyridine
(99 %), and sodium hydrosulfite (Certified ACS), ethylene gly-
col (purified, suitable for histological use) and 2-propanol (ACS
Plus) were obtained from Fisher. Nitrogen gas, (ultra high puri-
ty), a 50:50 nitrogen:oxygen gas mixture (reagent grade), and
oxygen gas (reagent grade) were obtained from Roberts Oxygen
Company. Glycerol (ultra-pure) was obtained from MP
Biomedicals, and dichloromethane (spectrophotometric
grade) was obtained from Acros. With the exception of
dichloromethane, which was dried, all reagents were used as
received. Hazardous waste was collected for disposal.
3d-d
3
L( )
3MLCT
M(t2g)
L( )
So
A
B
Fig. 1 Simplified orbital diagram a and lowest triplet state orderings b
for [Os(L)(L)(L)]2+ where L = α-diimine ligands such as bipyridine,
phenanthroline, and 4,7-diphenyl-1,10-phenanthroline [20, 30]
energy gap law [4, 20]. Modifying ligands to increase the
MLCT slightly can thus increase the luminescence intensity of
osmium complexes by raising the energy of the MLCTstate [4].
In the current work, maximum emission wavelength as well
as lifetime and luminescence anisotropy were tuned based on
the identity of the ligands and their influence on the MLCT state
and the degree of rotational diffusion. In order to elucidate
photophysical trends including the emission wavelength
and impact of hydrodynamic radius and net dipole mo-
ment on luminescence anisotropy, ligands were system-
atically altered in a set of homologous diimine osmium (II)
complexes, as shown in Fig. 2. All complexes contain the
bidentate bathophenanthrolinedisulfonic acid disodium salt
ligand (sulf-dpp) to ensure water solubility for applications
in hydrogel-based sensing. The addition of 2,2′-bipyridine
(bpy), 1,10-phenanthroline (phen), or 4,7-diphenyl-
phenanthroline (dpp) allowed for tuning of the MLCT state
as well as the inversely related hydrodynamic radius and net
dipole moment. The addition of strong crystal field carbonyl
ligands was expected to increase the MLCT above that of the
tris-substituted polypyridyl complexes, while also allowing
the impact of increased dipole moment on luminescence an-
isotropy to be assessed. Observed trends in photophysical be-
havior are rationalized, and the potential for this family of
complexes in hydrogel-based sensors is illustrated through
use of two of the complexes in a hydrogel-based relative hu-
midity sensor.
Synthesis of [Os(Phen)3]Cl2 Ammonium hexachloroosmate
and 1,10-phenanthroline in a 1:4 M ratio were refluxed in
ethylene glycol for 2 h under nitrogen according to literature
methods [32, 33]. A color change from burgundy to brown to
black was observed. [Os(phen)3]Cl2 was extracted in a 4:1
ethyl ether:acetone solution (×5), and the dark gray solid
was recovered by rotary evaporation in 70 % yield. A single
exponential fit to the excited state lifetime decay indicated that
no structurally similar luminescent impurities were present.
Synthesis of [Os(L)2(Sulf-Dpp)]Cl2 The preparation of the
homologous series of osmium complexes with alpha-
diimine ligands was modified from previous literature
methods [5]. The precursor Os(L)2Cl2 was synthesized by
refluxing ammonium hexachloroosmate in a 1:2 M ratio with
L = 2,2″-bipyridine (bpy), 1,10-phenanthroline (phen), or
bathophenanthroline (dpp) in 10 mL of ethylene glycol under
nitrogen for 45 min. After cooling to room temperature, sodi-
um hydrosulfite (0.17 M) was added to reduce the osmium
from Os(IV) to Os(II). Precipitation of Os(bpy)2Cl2,
Os(phen)2Cl2, and Os(dpp)2Cl2 was achieved via an ice-bath,
and the solid was washed with deionized water and ethyl
ether. Yields were close to 80 %.
Os(L)2Cl2 and bathophenanthrolinedisulfonic acid
disodium salt hydrate (sulf-dpp) were combined in a 1:1 M
ratio with 15 mL of 60:40 solution of ethanol:water and
refluxed under nitrogen for 5 h. After full conversion as de-
termined by TLC, the product, [Os(bpy)2(sulf-dpp)]Cl2,
[Os(phen)2(sulf-dpp)]Cl2, or [Os(dpp)2(sulf-dpp)]Cl2, was
precipitated in acetone in 35 % yield. Conversion was sup-
ported by the disappearance of the diagnostic IR peak at
Experimental
Materials Bathophenanthrolinedisulfonic acid disodium salt
hydrate (98 %), ammonium hexachloroosmate (IV) (99.9 %),

J Fluoresc
760 cm−1 corresponding to chloride ligands and the appear-
ance of a peak at 1190 cm−1 indicating the presence of the
sulfonated ligand. A single exponential fit to the excited state
lifetime decay indicated that no structurally similar lumines-
cent impurities were present. [Os(bpy)2(sulf-dpp)]Cl2 1H
NMR (400 MHz, D2O): δ = 2.66 [d, 2H, Ar-H], 8.34 [m,
4H, Ar-H], 7.82 [d, 2H, Ar-H], 7.71 [m, 4H, Ar-H], 7.62 [d,
2H, Ar-H], 7.44 [br, 2H, Ar-H], 7.12–7.30 [m, 10H, Ar-H],
6.85 [br, 2H, Ar-H]. IR (KBr pellet) υ(cm−1): 3448(m),
1638(m), 1394(s), 1194(s), 1125(m), 1034(m), 795(w).
[Os(phen)2(sulf-dpp)]Cl2:1H NMR (400 MHz, D2O):
δ = 8.88 [d, 2H, Ar-H], 7.88 [br, 2H, Ar-H], 7.74[[m, 4H,
Ar-H], 7.31–7.56 [m, 12H, Ar-H], 7.108[t, 2H, Ar-H],
7.01[br, 2H, Ar-H], 6.97 [br, 2H, Ar-H], 6.84[br, 2H, ar-H].
IR (KBr pellet) υ(cm−1): 3423(m), 2995(w), 1605(w),
1560(m), 1485(w), 1425(s), 1406(s), 1384(s), 1275(m),
1192(s), 841(s), 733(m), 711(m). [Os(dpp)2(sulf-dpp)]Cl2:1H
NMR (400 MHz, D2O): δ = 9.04 [br, 6H, Ar-H], 7.81–7.87
[m, 20H, Ar-H], 7.72 [br, 6H, Ar-H], 7.63 [m, 14H, Ar-H]. IR
(neat) υ(cm−1): 3448(w), 2997(w), 1619(w), 1572(w),
1430(m), 1416(m), 1396(m), 1226(s), 1181(m), 1075(s),
1026(m), 910(m), 845(s), 828(s), 761(m), 700(s).
by refluxing 0.95 mmol ammonium hexachloroosmate with
15 mL of formaldehyde in 40 mL formic acid for three days.
The solution progressed in color from red to brown to yellow.
The solvent was removed in vacuo, and the product was
titurated with dicholoromethane and collected by filtration.
The product was dissolved in acetone, NH4Cl was removed
by filtration, and the solvent was removed in vacuo, resulting
in a light brown solid of [Os(CO)2Cl2] in 50 % yield.
[Os(CO)2Cl2(sulf-dpp)] was synthesized by refluxing
bathophenanthrolinedisulfonic acid disodium salt hydrate
and [Os(CO)2Cl2] in a 3:2 M ratio for 8 h in 15 mL 2-
propanol with ~2 mL water added for increased solubility.
Conversion to the final product was supported by the decrease
in signal of diagnostic IR peaks at 2122 cm−1 and 2012 cm−1
corresponding [Os(CO)2Cl2] and the growth of IR peaks at
2136 cm−1, 2037 cm−1, and 1960 cm−1 corresponding to
[Os(CO)2Cl2(sulf-dpp)]. [25] The solvent was removed in
vacuo, and a Sephadex G-50 column with methanol eluent
was used to purify [Os(CO)2Cl2(sulf-dpp)] for a 50 % yield.
1H NMR (400 MHz, D2O): δ = 7.34–7.55 [m, 8H, Ar-H], 7.75
[d, 2H, Ar-H], 7.80 [d, 2H, Ar-H], 8.19 [d, 2H. Ar-H]. IR
(KBr pellet) υ(cm−1): 3422(m), 2959(w), 2136(m), 2037(m),
1961(m), 1617(m), 1384(s), 1194(m), 115(w), 1032(m).
Synthesis of [Os(CO)2Cl2(Sulf-Dpp)] The synthesis of
[Os(CO)2Cl2(sulf-dpp)] was modified from a published pro-
cedure [34, 35]. The precursor [Os(CO)2Cl2] was synthesized
Photophysical Characterization The photophysical proper-
ties of the complexes in deionized water, glycerol, and
Fig. 2 Water-soluble osmium
(II) complexes with
polypyridyl ligands

J Fluoresc
1.2
1
0.35
0.3
1.2
1
0.25
0.2
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.15
0.1
0.05
0
200
400
600
800
300
500
700
Wavelength (nm)
Wavelength (nm)
Fig. 4 Normalized excitation (- - -)and emission spectra (-.- excited at
395 nm; — excited at 500 nm) with anisotropy spectra (△ emission at
550 nm; ○ emission at 730 nm; *excitation at 395 nm; x excitation at 500
nm) for [Os(CO)2Cl2(sulf-dpp)] in glycerol
Fig. 3 Normalized absorbance (- - -), excitation (…), and emission
spectra (—) of [Os(bpy)2(sulf-dpp)]Cl2 in water at room temperature
glycerol:water solutions of varying viscosity were measured
at room temperature. A Shimadzu recording UV-VIS spectro-
photometer (UV-2401PC) was used to obtain absorbance
spectra, and a Fluorolog Horiba Jobin Yvon Fluorolog-3
Model FL3–22 spectrofluorometer was used to obtain uncor-
rected and corrected luminescence excitation and emission
spectra, steady state luminescence anisotropy, and excited
state lifetime. For spectral acquisition, polarizing filters were
used in the excitation and emission paths at 0° and 90°, re-
spectively, in order to minimize contributions from scattered
radiation. Anisotropy measurements (8 replicates for spectra
and 100 replicates for single wavelength analysis) and lifetime
measurements (in triplicate) were acquired using the uncor-
rected excitation and emission maxima. Excited state lifetimes
were acquired via TCSPC using the Horiba Fluorohub-B ac-
cessory and DAS6 Fluorescence-Decay Analysis Software; a
450 nm Horiba Scientific NanoLED laser was the source.
Four-point Stern-Volmer oxygen quenching plots were ob-
tained from the excited state lifetime measurements (in tripli-
cate) of nitrogen-, air-, 50:50 nitrogen:air-, and oxygen-
saturated solutions.
a saturated potassium persulfate solution. After stirring for
1 min, 400 uL of the prepolymer solution was delivered via
microliter pipet onto a pre-scored, glass microscope slide
(Corning, 7.6 cm × 2.5 cm × 1 mm), spread uniformly over
the surface, and allowed to polymerize at room temperature
for 24 h before heating in an oven at 55 °C for 2 h. Six
2.5 cm × 0.8 cm sensing films were obtained from each mi-
croscope slide. The response of resultant sensor films to
changing relative humidity was calibrated in flowing gas as
described previously [1]. Humidity was varied by controlling
the relative flow rates from two nitrogen gas tanks—one dry
and one sent through a deionized water bubbler—which were
passed into an airtight mixing chamber. Relative humidity was
measured at ambient temperature using a Vernier RH-BTA
relative humidity probe placed in the mixing chamber just
prior to an outlet tube that was directed into the cuvette con-
taining the sensor film in the spectrofluorimeter. Sensor films
were preconditioned by cycling back and forth between 10
and 100 % humidity until constant emission response, mea-
sured from the front face, was observed.
Preparation and Calibration of Humidity Sensor The
prepolymer solution was prepared as described previously
[1] by adding 0.5 mg of [Os(bpy)2(sulf-dpp)]Cl2 to 1.3 mL
of 40 wt.% acrylamide solution, 1.2 mL of 2 wt.% N-N′-
methylenebisacrylamide solution, and 0.4 mL of 2-(dimethyl
amino)ethylmethacrylate. The solution was purged under ni-
trogen with stirring for 20 min prior to the addition of 20 uL of
Results and Discussion
As shown in Fig. 3, the absorption spectrum of
[Os(bpy)2(sulf-dpp)]Cl2 in water at room temperature exhibits
Table 1 Maximum absorption
and corrected emission
wavelengths of osmium
complexes
Maximum absorption
wavelength (nm)
Corrected maximum emission
wavelength (nm)
Water
Glycerol
Water
760
750
745
730
725
Glycerol
750
[Os(bpy)2(sulf-dpp)]Cl2
[Os(phen)2(sulf-dpp)]Cl2
[Os(dpp)2(sulf-dpp)]Cl2
[Os(CO)2Cl2(sulf-dpp)]
[Os(phen)3]Cl2
435, 480
435, 480
450, 495
445, 490
430, 475
440, 485
440, 480
450, 495
450, 495
435, 480
740
740
570, 730
735

J Fluoresc
an intense π-π* ligand-centered absorption and a double-
humped MLCT band with maxima at 435 and 480 nm. The
absorption peak exhibits a tail extending to 700 nm, thus
allowing for the use of low-cost excitation sources. Spectra
obtained for [Os(phen)2(sulf-dpp)]Cl2 and [Os(dpp)2(sulf-
dpp)]Cl2 showed similar peak shapes with maxima as reported
in Table 1 and an extended tail to 700 nm. The observed peak
shape is consistent with that obtained in the current work and as
previously reported in the literature for [Os(phen)3]Cl2 [22, 33].
Excitation spectra for [Os(bpy)2(sulf-dpp)]Cl2 (Fig. 1) and
its analogs exhibit a broad MLCT band with the maximum
excitation wavelength corresponding to the shorter wave-
length MLCT absorption maximum. In the excitation spectra,
the contribution of the 315 nm π-π* ligand-centered peak was
more pronounced for these complexes containing the sulfonat-
ed dpp ligand as compared to [Os(phen)3]Cl2 for which the
ligand-centered excitation peak was of significantly lower in-
tensity. The relative magnitude of the ligand-centered peak
was found to increase from bpy to phen to dpp in the homol-
ogous series, consistent with the increasing conjugation of the
ligands and stabilization of the excited state.
Wavelengths of maximum emission for the series of
[Os(L)2(sulf-dpp)]Cl2 complexes in aqueous solution
(Table 1) decreased from L = bpy to phen to dpp (760, 750,
and 745 nm, respectively), indicating an increase in energy of
the MLCT excited state resulting from the increase in ligand
field strength. Further evidence of the relationship between
strength of pi-acceptors and emission wavelength is apparent
in the comparison of [Os(sulf-dpp)3]Cl2, which was previous-
ly reported in the literature, to [Os(dpp)2(sulf-dpp)]Cl2. The
addition of sulfonated groups should enhance pi-accepting
ability of the dpp ligand, and indeed, the tri-substituted sulf-
dpp complex exhibited a shorter emission wavelength
(731 nm for the complex in 1 M tris HCl, pH 7) [22, 33] than
the mono-substituted sulf-dpp complex. When comparing
[Os(phen)3]Cl2 to [Os(phen)2(sulf-dpp)]Cl2, it seems apparent
that the decrease in energy of the MLCTstate is a consequence
of the changing symmetry of the complex as opposed to the
addition of a sulfonated dpp ligand.
emission wavelengths of 570 and 730 nm, measured optimal-
ly with 390 nm and 450 nm excitation, respectively. The ab-
sorption spectrum was dominated largely by ligand-localized
transitions between 250 and 350 nm, but a small MLCT side-
band was observed to extend to 650 nm. The higher energy
emission is believed to originate from a charge transfer excited
state localized on the less readily reduced carbonyl ligands,
while the lower energy emission, which is consistent with
results obtained for complexes containing alpha-diimine li-
gands (Table 1), is believed to originate from a charge transfer
excited state localized on the sulf-dpp ligand. Both excited
states are too low in energy to be attributed to ligand-
localized π➔π* excited states, given that the free sulf-dpp
ligand exhibits optimum excitation and emission wavelengths
of 340 nm and 390 nm, respectively. Not surprisingly, emis-
sion from the higher energy excited state is not observed for
the complex in aqueous solution, likely a consequence of rap-
id internal conversion to the lower energy charge transfer
state. Also as expected, the addition of the carbonyl served
to increase the energy of the excited state and thus decrease the
maximum emission wavelength as compared to the tris
polypyridyl complexes (Table 1). Despite the presence of
chloride ligands, constant emission intensities indicated that
photodecomposition was not a significant issue, likely a con-
sequence of the use of solvents of high dielectric constant and
the presence of the alpha diimine ligand [36, 37].
It was anticipated that the excited state lifetimes of the
osmium complexes would follow the energy gap law which
suggests that excited state lifetimes and quantum yields within
a homologous series will decrease exponentially as the MLCT
energy is lower and nonradiative processes compete more
effectively [4, 22, 38–40]. The bpy complex with the longest
emission wavelength indeed exhibited the shortest excited
state lifetime, as observed in Table 2 for nitrogen-purged so-
lutions. Slightly longer excited state lifetimes were observed
for the phen, dpp, and carbonyl-containing complexes, con-
sistent with their shorter emission wavelengths, but the differ-
ence between them is not statistically significant. Likewise,
the compounds were equally emissive, exhibiting similar
quantum yields that are at least an order of magnitude smaller
than [Ru(bpy)3]Cl2 [39, 41] but appreciable such that small
As shown in Fig. 4, [Os(CO)2Cl2(sulf-dpp)] measured in
glycerol exhibited two distinct excited states with maximum
Table 2 Excited state lifetimes, Stern-Volmer oxygen quenching constants, and bimolecular quenching rate constants for osmium complexes in
aqueous solution
N2 (ns)
Air (ns)
50:50 N2:O2 (ns)
O2 (ns)
KSV (M−1
)
kO (M−1 s−1
)
R2
[Os(bpy)2(sulf-dpp)]Cl2
[Os(phen)2(sulf-dpp)]Cl2
[Os(dpp)2(sulf-dpp)]Cl2
[Os(CO)2Cl2(sulf-dpp)]
[Os(phen)3]Cl2
48
74
75
73
78
3
4
4
5
3
47
73
71
69
73
2
5
6
3
4
43
65
56
63
62
4
5
8
4
5
38
56
49
55
52
3
4
5
5
5
6.7 0.6
8.3 0.1
(1.4 0.2) × 108
(1.1 0.2) × 108
(1.9 0.3) × 108
(1.1 0.1) × 108
(1.6 0.1) × 108
0.984
0.974
0.958
0.999
0.993
14
8.1 0.2
12.6 0.8
2
*uncertainties reported as 95 % C. L

J Fluoresc
1.2
1
0.2
amounts of compound give reasonable signal for the
ratiometric types of sensors described here.
Stern-Volmer oxygen quenching constants, KSV, were ob-
tained from four-point Stern-Volmer plots generated from the
excited state lifetimes in Table 2 and the Stern-Volmer
Eq. (Equation 2)
0.15
0.1
0.8
0.6
0.4
0.2
0
0.05
0
-0.05
-0.1
-0.15
-0.2
τo
¼ 1 þ KSV ½O2Š
ð2Þ
τ
200
700
Wavelength (nm)
where τo and τ are the excited state lifetime in the absence and
presence of oxygen, respectively. Bimolecular quenching rate
constants, ko, reported in Table 2 were calculated from the
Stern-Volmer quenching constant, which is the product of
the biomolecular quenching rate constant and τo [42]. As ex-
pected for dynamic quenching in solution, plots were highly
linear, indicated by the R-squared value. Measured Stern-
Volmer quenching constants were quite low as compared to
a value of 2130 M−1 for the well-utilized oxygen sensor
[Ru(bpy)3]2+ [41], which makes these osmium complexes ad-
vantageous in applications where oxygen concentrations are
variable. The bimolecular quenching rates constants are with-
in experimental error of one another as expected because the
accessibility of the luminophores to the quencher is expected
to be roughly the same for this family of complexes [42].
Excited state lifetimes of air-equilibrated aqueous solutions
of the tris-substituted polpyridyl complexes exhibit a depen-
dence on the viscosity of the solution and thus rigidity of the
local environment of the luminophores, as seen in Table 3. The
decrease in excited state lifetime with increasing glycerol con-
centration observed for complexes in Table 3 is too significant
to be attributed solely to a reduction in the Henry’s Law solu-
bility of oxygen in glycerol relative to water, particularly given
the low quenching constants for these complexes. The low
intensity of [Os(CO)2Cl2(sulf-dpp)] in glycerol-containing sol-
vents precluded accurate measurement of excited state lifetimes
for comparison. With its low Stern-Volmer quenching constant
and good environmental sensitivity, [Os(bpy)2(sulf-dpp)]Cl2
shows promise as a reporter molecule in hydrogel-based sen-
sors with excited state lifetime as the means of analyte mea-
surement. Shorter excited state lifetimes would be expected as
the hydrogel swells in response to a change in analyte
concentration.
Fig. 5 Excitation (dotted) and emission (solid) spectra with anisotropy
for [Os(bpy)2(sulf-dpp)]Cl2 in glycerol
The luminescence anisotropy of the homologous series of
osmium complexes was also investigated to determine wheth-
er hydrodynamic radius (i.e., size) or net dipole moment has a
greater impact. Anisotropy with respect to excitation and
emission are shown in Figs. 4 and 5 for [Os(CO)2Cl2(sulf-
dpp)] and [Os(bpy)2(sulf-dpp)]Cl2 measured in glycerol. As
expected, the emission anisotropy is independent of wave-
length for [Os(bpy)2(sulf-dpp)]Cl2 because the emission orig-
inates from the lowest energy excited state [19]. In the case of
[Os(CO)2Cl2(sulf-dpp)], the emission anisotropy shows a de-
pendence on wavelength because of the population of the two
charge transfer excited states, but, as expected, the anisotropy
is constant for a given excited state with the higher energy
excited state exhibiting greater anisotropy (Fig. 4). For both
complexes, there is an increase in excitation anisotropy with
increasing wavelength of excitation.
Table 4 summarizes anisotropy measurements in glycerol
and 6:4 glycerol:water solutions for the family of complexes.
Not surprisingly, anisotropy decreased significantly upon the
addition of water since the molecules rotate more freely in
solution. The measured anisotropy values in 6:4 glyercol:water
are within experimental of one another. In glycerol, the magni-
tude of the luminescence anisotropy is observed to decrease for
the complexes from substitution of bpy to phen to dpp. This
inverse relationship between anisotropy and hydrodynamic ra-
dius suggests that dipole moment has the greater effect. Further
support of the dependence on dipole moment is evidenced by
the high anisotropy of the carbonyl-containing complex and the
low anisotropy of the [Os(phen)3]Cl2 complex. The highest
Table 3 Excited state lifetimes of
air-equilibrated aqueous solutions
of osmium complexes in water
and glycerol solutions
Excited state lifetime (ns)
H2O
6:4 Glycerol:H2O
9:1 Glycerol:H2O
Glycerol
of varying viscosity
[Os(bpy)2(sulf-dpp)]Cl2
47
73
71
2
5
6
81
5
100
150
153
5
5
5
133
179
178
8
5
3
[Os(phen)2(sulf-dpp)]Cl2
[Os(dpp)2(sulf-dpp)]Cl2
117
112
5
5
*uncertainties reported as 95 % C. L

J Fluoresc
Table 4 Luminescence anisotropy at optimized excitation and emission wavelengths of osmium complexes at 298 K
6:4 Glycerol: water solutions
Glycerol solutions
λ (nm)a Ex/Em
500/750
Luminescence anisotropy
λ (nm)a Ex/Em
500/750
Luminescence anisotropy
0.071 0.004
[Os(bpy)2(sulf-dpp)]Cl2
[Os(phen)2(sulf-dpp)]Cl2
[Os(dpp)2(sulf-dpp)]Cl2
[Os(CO)2Cl2(sulf-dpp)]
0.005 0.003
0.005 0.004
0.004 0.004
0.010 0.004
510/740
510/740
0.054 0.006
525/740
525/740
0.031 0.008
490/730
490/730
0.080 0.010
500/550
0.277 0.001
[Os(phen)3]Cl2
430/725
0.000
430/725
0.028 0.003
a Optimum excitation and emission wavelengths were determined from anisotropy versus excitation and anisotropy versus emission spectra for each
complex
*Uncertainties reported as 95 % C. L
anisotropy overall is observed for [Os(CO)2Cl2(sulf-dpp)]
when excited at 500 nm, thus suggesting that this as the most
useful luminophore for application in hydrogel-based sensors
where a change in anisotropy would be expected upon swelling
and contracting of the hydrogel in response to analyte.
wavelength-based sensor reported previously, which exhibited
only a 15 nm wavelength shift over the full humidity range. [1]
Figure 7, which shows results obtained for a sensor film
containing [Os(CO)2Cl2(sulf-dpp)] as the reporter molecule il-
lustrates several important points. Most significantly, the anisot-
ropy is observed to decrease with increasing relative humidity.
This is a consequence of the fact that anisotropy was monitored
for the higher energy charge transfer excited state which is
likely to be more highly populated when the hydrogel is
contracted and the complex is in a rigid environment. There is
no change to anisotropy until the relative humidity reaches
about 40 %, suggesting that this is the point at which internal
conversion to the lower energy charge transfer state begins to
play a significant role. While dynamic range is thus limited for
this particular sensor to between 40 and 100 % RH, it seems
likely that through optimization of hydrogel composition and,
more specifically, pore size, it should be possible to control
dynamic range. Error bars obtained in Fig. 7 are sizable, as is
the case for most anisotropy calibrations obtained for a single
hydrogel sensing film of this type. Poor precision is attributed,
in part, to the heterogeneity of the reporter molecule within the
sensor films. A similar effect is observed for luminescence-
In order to demonstrate proof-of-concept for use in
hydrogel-based sensors, [Os(bpy)2(sulf-dpp)]Cl2 and
[Os(CO)2Cl2(sulf-dpp)] were embedded in hydrogel films
for use as humidity sensors similar to those reported previously
with dapoxyl sulfonic acid as the environment-sensitive report-
er molecule. [1] Because anisotropy was observed to change
more appreciably than excited state lifetime with changing ri-
gidity of the local environment of these luminophores, lumi-
nescence anisotropy was monitored as a function of percent
relative humidity. Figure 6 shows pooled anisotropy measure-
ments for three independently prepared sensing films contain-
ing [Os(bpy)2(sulf-dpp)]Cl2. Film-to-film reproducibility was
reasonable, and it was observed that as the hydrogel swelled
in response to an increase in relative humidity, there was a
concomitant increase in measured anisotropy. This is attributed
to the increased solvation and thus decreased rotational diffu-
sion of the reporter molecule with swelling of the hydrogel and
uptake of water vapor. The 1.3-fold change in anisotropy
shown in Fig. 6 is superior to the sensitivity of our
Increasing RH
Decreasing RH
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.105
0.100
0.095
0.090
0.085
0.080
0.075
0.070
0
50
100
0
50
Relative Humidity (%)
100
Relative Humidity (%)
Fig. 6 Pooled anisotropy response as a function of percent relative
humidity for three acrylamide/2-(dimethyl amino)ethylmethacrylate
sensing films with [Os(bpy)2(sulf-dpp)]2+ as the reporter molecule
Fig. 7 Anisotropy as a function of percent relative humidity for an
acrylamide/2-(dimethyl amino)ethylmethacrylate sensing film with
[Os(CO)2Cl2(sulf-dpp)] as the reporter molecule

J Fluoresc
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Conclusion
The osmium complexes reported here, and particularly
[Os(bpy)2(sulf-dpp)]Cl2 and [Os(CO)2Cl2(sulf-dpp)], show
great promise as reporter molecules in hydrogel-based sensors
or any sensing scheme requiring a change in emission re-
sponse upon change in local environment of the reporter mol-
ecule. Distinct advantages include their water solubility, the
ability to make use of longer wavelength excitation as com-
pared to their ruthenium and rhenium counterparts, and the
opportunity to eliminate sources of error typically associated
with intensity-based measurements through use of ratiometric
anisotropy or intensity-independent excited state lifetime mea-
surements. In addition, the tailorability of the osmium com-
plexes in terms of hydrodynamic radius, dipole moment, and
photophysical properties should allow further optimization for
particular sensing applications with a given support matrix.
Likely, greatest sensitivity would be achieved in a hydrogel-
based sensor when the pore size in the contracted hydrogel is
closely matched to the hydrodynamic radius of the complex.
Under such circumstances, the complex would experience a
largely hydrophobic environment when the hydrogel is
contracted, and even a very small degree of swelling should
result in a significant change in the polarity of the local envi-
ronment. As the results here have shown, it is possible to
control the dynamic range of a sensor by varying the reporter
molecule, thus providing a means by which to increase sensi-
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Acknowledgments The authors wish to acknowledge Kayla A. Hess
for her scholarly contributions and the Elizabethtown College E. Jane
Valas Undergraduate Research Fund and Scholarship and Creative Arts
Program for financial support.
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