Mechanisms of Photochemistry and
Reactive Oxygen Production by
Fullerene Suspensions in Water
E R N E S T M . H O T Z E ,†J E R O M E L A B I L L E ,‡
P E D R O A L V A R E Z ,§A N D
M A R K R . W I E S N E R *, †
Department of Civil and Environmental Engineering, Duke
University, Durham, North Carolina 27708-0287, CEREGE,
University Aix-Marseille, Aix-en-Provence, France, and
Department of Civil and Environmental Engineering, Rice
University, Houston, Texas 77251-1892
Received August 29, 2007. Revised manuscript received
January 13, 2008. Accepted March 12, 2008.
Buckminsterfullerene (C60) is a known photosensitizer that
produces reactive oxygen species (ROS) in the presence of
light; however, its properties in aqueous environments are still
not well understood or modeled. In this study, production of
both singlet oxygen and superoxide by UV photosensitization of
colloidal aggregates of C60in water was measured by two
distinct methods: electron paramagnetic resonance (EPR) with
a spin trapping compound, and spectrophotometric detection
while neither was detected in the aqu/nC60suspensions. A
mechanistic framework for photosensitization that takes into
to explain these results. While theory developed for single
molecules suggests that alterations to the C60cage should
reduce the quantum yield for the triplet state and associated
ROS production, the failure to detect ROS production by aqu/
nC60is explained in part by a more dense aggregate structure
compared with the hydroxylated C60.
Commercial products containing fullerenes are already on
the market. These include tennis racquets (1), epidermal
growth factor (2), and facial antioxidant cream (3) to name
a few. The availability and use of these products and a
substantial increase in production forecasted for fullerenes
suggests that these materials may make their way into
wastewater treatment influents and aquatic environments.
number of fullerene variations through functionalization.
Also, the behavior of these materials may be altered by the
However, an evaluation of the surface and photochemistry
of some relatively simple aqueous suspensions of fullerenes
impacts of fullerenes and basis for benchmarking more
The properties of C60 and other fullerenes have been
described in numerous studies with respect to fullerene
toxicity (4–8), antioxidant capacity (9–11), and characteriza-
tion (12–15). The ability of C60to produce reactive oxygen
species, or ROS, (e.g., (16)) has received considerable
attention, in part based on the implications for toxicity to
cells (5) in both medical and environmental contexts as well
as the potential applications implied for industrial or water
treatment technologies (17, 18). However, unlike the condi-
tions leading to ROS production via photosensitization by
individual C60 molecules in organic solvent, (19, 20), the
conditions that lead to ROS production by C60in water and
the reactive species formed are less well understood. We
have previously reported on the ability of hydroxylated C60
(fullerol) to produce ROS in water (17, 18), but have not
Photosensitized molecules like fullerene are capable of
transferring light energy to chemical oxidation potential in
energy directly to an oxygen molecule, ROS formation may
occur via a type II pathway primarily resulting in singlet
oxygen (1O2). Type I ROS formation occurs when photosen-
sitization increases interactions between a photosensitizer
molecule and an electron donor, ultimately leading to the
transfer of an electron to an oxygen and the production of
radicals such as superoxide (O2-•). The efficiency of light
be expressed as a quantum yield (21). Pristine fullerene
suspended in a nonpolar solvent has a quantum yield near
photosensitization and formation of its triplet excited state
(3C60). However the introduction of fullerenes into a polar
solvent, such as water, results in a very different chemical
environment and physical configuration of C60 compared
with the organic solutions of C60that have been the subject
of previous studies of ROS formation. In particular, stable
suspensions of fullerenes in water tend to be present as
fullerenes in water is complicated by the low solubility of
many of these materials in water. Nonetheless, stable
suspensions of these materials may be produced either
intentionally or naturally through encapsulation (22–25),
functionalization (26–28), or aggregation (29–32).
somewhat less due to steric hindrance, and poly function-
alized fullerenes exhibit greater stability with respect to
limited aggregation. Nonetheless, aggregation of poly func-
tionalized C60may readily occur and has been observed in
some cases to increase with concentration (15), whereas in
other cases, concentration does not seem to have an effect
on aggregation (35). One of the more studied poly func-
which can have a varying amount of hydroxyl groups on its
surface depending on reaction conditions (26). Even with
in suspensions that are stable to a maximum concentration
of approximately 38.5 mM (15), depending on the number
of hydroxyl groups added.
Stable colloidal suspensions of initially unfunctionalized
C60can be made by extended stirring (25, 31) or sonication
in water (aqu/nC60) (36) or through solvent exchange using
(37). However, these latter colloidal suspensions of C60may
* Corresponding author phone: 919-660-5292; fax: 919-660-5219;
Environ. Sci. Technol. 2008, 42, 4175–4180
10.1021/es702172w CCC: $40.75
Published on Web 04/29/2008
2008 American Chemical SocietyVOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94175
contain residual quantities of the organic solvent (13).
Differences in the aggregation state of C60 as well as
may affect the ability of suspensions of these aggregates to
produce ROS (38). One premise examined in this paper is
that a higher proximity of two C60 cages within a dense
aggregate may decrease ROS production by increasing the
likelihood of processes such as triplet–triplet annihilation
less of the fullerene to the solution, potentially reducing the
active surface area for ROS production. In this study, we
compare ROS production by two colloidal suspensions of
C60having very different aggregate structures as quantified
by X-ray diffraction, aqu/nC60 and fullerol. We further
differentiate conditions favoring type I and type II photo-
sensitization reactions by these two suspensions.
2. Materials and Methods
were purchased from MER (Tucson, AZ). Superoxide dis-
1-oxide (DMPO), 2,2,6,6-tetramethyl-4-piperidinol (4-oxo-
TEMPO), Adenosine 5′-(trihydrogen diphosphate) (NADH),
2,2,6,6-tetramethyl-4-piperidone (TEMP), ?-carotene, and
Louis, MO). Deuterium Oxide (D2O) was purchased from
Cambridge Isotope Laboratories (Andover, MA). Ultrapure
water had resistivity greater than 10 MΩ cm and dissolved
organic carbon concentration <3 µg/L.
and medium pressure UV lamps. Low pressure (LP) UV
output spectrum peak at 365 nm and a total irradiance of
by a low pressure (LP) UV light source was necessary for
proximity to the EPR equipment.
Medium pressure (MP) irradiation (Calgon Carbon Cor-
poration Pittsburgh, PA) was provided by a bench scale
collimated beam (39). These experiments were carried out
in a Petri dish with known sample depth and surface area
allowing for a calculation of UV fluence (mJ/cm (2)). A UV
radiometer and detector (International Light Inc., model
1700/SED 240/W) calibrated at 2 nm intervals in the range
of 200-400 nm was used to measure UV irradiance at the
top of the suspension. UV fluence (mJ/cm2) was calculated
as the average irradiance multiplied by the exposure time.
The average UV irradiance in the completely mixed sample
and sample depth using an integrated form of the Beer–
Lambert law. Utilizing a shutter, samples of various con-
centrations were exposed to UV light for 5 minute time
intervals under the broadband MP UV source, the fluence
was calculated as the total UV output in the 200-300 nm
region. UV fluence was used as a normalizing factor in
determining the capacity of each suspension to produce
superoxide radicals. It should be noted that light under 300
nm in wavelength is more relevant to engineered systems
rather than natural systems. Wavelengths larger than 300
nm are likely to be less effective in photosensitization. Our
goal here, however, is to explore mechanisms of ROS
is needed to determine the rates of ROS that would occur in
natural systems under typical solar illumination.
for all measurements were: frequency, 9.27 GHz; power, 5
kHz. UV/vis spectrometry was preformed using a Hitachi
U-2000 spectrophotometer. X-ray diffraction (XRD) was
preformed using an X’Pert-Pro diffractometer from PANa-
lytical instruments equipped with a Co anode source, and
an RTMS scanning detector was used. Dynamic light scat-
tering was preformed using a Zetasizer nano ZS (Malvern
laser (633 nm) and collects time variable scattering data at
a fixed angle of 173°.
2.4. XRD Procedure. The structure of the C60molecules
X-ray scattering by samples prepared by depositing a few
drops of aqu/nC60or fullerol suspension on a silicon plate
and allowing the water to evaporate. The 2 theta angle was
step, and the divergence slit was automated so that a 10 mm
sample length was constantly irradiated.
was used to trap singlet oxygen. TEMP has a low detection
limit and a relatively long adduct lifetime when compared
with other spin traps. This makes it an ideal trap for
A mixture of the fullerene colloidal suspension and 80 mM
into a sample tray for UV irradiation. Upon removal, the
sample was taken up in a capillary tube, capped with clay,
holder. In each case the EPR parameters were held constant
as was the TEMP concentration. Separate samples were
irradiated under UV for up to 3 h. Signals were compared
with the standard product of TEMP and singlet oxygen
(TEMPO) in order to determine singlet oxygen generation
2.6. Superoxide Concentration Analysis by XTT. XTT
reduction was employed to measure the production of
superoxide. The reduction of XTT results in an increase in
optical density at 470 nm that can be used to quantify the
relative amount of superoxide present (43, 44). The con-
reduction with and without a quencher for superoxide,
related XTT reactions to be accounted for. Samples were
prepared by mixing 10 mL flasks with the appropriate
suspension and 100 µM XTT. One set of experiments was
to the samples to quench any superoxide generated. In
experiments irradiated by the MP UV lamp, flasks were
poured into a Petri dish with surface area approximately 40
of suspensions at 470 nm. While the physical chemical
properties of XTT and TEMP do not favor adsorption to
fullerene surfaces, possible interference arising from XTT or
TEMP adsorption to aqu/nC60 or fullerol aggregates was
addressed by adding the compounds in excess as calculated
from available fullerene surface area and KOWrelationships
(see Supporting Information).
2.7. Suspension Preparation. Aqu/nC60 was prepared
from a supersaturated suspension of C60 in water (DDW).
Here approximately 0.4 mg/mL of powdered C60was added
to DDW. The solution was then stirred and sonicated for
approximately 6 h with a Branson sonifier ultrasonic cell
characteristic of nC60suspensions, and was filtered through
a 0.45 µm methyl cellulose ester (MCE) filter (to remove
unsuspended C60) and stored in the dark. The aqu/nC60was
also prepared by the same method in D2O. The final
4176 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 11, 2008
suspension was found to be stable for periods up to at least
light scattering (DLS). In both D2O and H2O, the mean
diameter of the aqu/nC60 was approximately 145 nm as
were made by adding approximately 0.07 mg/mL powdered
fullerol to DDW or D2O. This suspension was made up in a
volumetric flask and placed in the sonication bath for 2 h.
colored as more fullerol was stabilized in the suspension.
Once removed from sonication, the suspension was filtered
through 0.45 µm MCE filters via vacuum to remove unsus-
with a mean diameter of 200 nm as determined by DLS and
a total organic carbon concentration of 90 mg/L. The stock
over a period of at least three months. UV/vis absorbance
spectra for fullerol and aqu/nC60 suspensions have been
previously reported (13, 45).
3. Results and Discussion
3.1. Singlet Oxygen Production by aqu/nC60. EPR mea-
surements were undertaken to compare the singlet oxygen
generation capacities of the two types of C60suspensions.
The aqu/nC60did not produce a signal for the TEMP-singlet
oxygen adduct after 60 min of LP UV irradiation, even when
these suspensions were made in D2O to increase detection
sensitivity (Figure 1A). These results suggest that aqu/nC60
does not participate in detectable type II photosensitization
1B: Electron Paramagnetic Resonance signal for photo-
40 µM and fullerol 40 µM were irradiated with LP UV light.
between 3270 and 3320 G.
1C: Electron Paramagnetic Resonance signal for 40 µM
was irradiated under LP UV light for 15, 30, and 60 min. The
field is measured between 3270 and 3320 G.
and the standard signal from 0.05µM TEMPO in Figure 1B
to be 0.032µM/min when 40µM Fullerol suspended in D2O.
water was also used to increase singlet oxygen sensitivity in
experiments with the fullerol suspensions. In contrast with
via the type II photosensitization pathway. The response
produced by the fullerol suspension was compared with a
sensitizing molecule (21). Figure 1B compares the signal
corresponding to singlet oxygen generation produced by a
solution of 40 µM Rose Bengal after 5 min LP UV irradiation
following 15 min LP UV irradiation. The Rose Bengal
fullerol. The amplitude of the 0.5 µM TEMPO adduct
approximately matches the fullerol signal, meaning that 40
µM fullerol produces approximately 0.5 µM singlet oxygen
after 15min of LP UV irradiation.
Singlet oxygen generation with increasing UV fluence is
illustrated in Figure 1C where a 40 µM suspension of fullerol
FIGURE 1. A: Electron Paramagnetic Resonance signal between 3270 and 3320 G for a 40 µM aqu/nC60suspended in D2O with 80 mM
TEMP and irradiated under LP UV for 60 min. The absence of a triplet signal indicates the absence of singlet oxygen generation.
VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4177
was illuminated under UV for 15, 30, and 60 min. From a
measurement of signal amplitude and a comparison with
the amplitude of the TEMPO standard in Figure 1B an
estimate of 0.032 µM/min is obtained for the singlet oxygen
generation rate by a 40 µM fullerol suspension under LP UV
Singlet oxygen production by fullerol was significant
enough to be detected in suspensions of conventional H2O,
but the signal is less distinct and 5 times lower in amplitude
for fullerol suspended in water because singlet oxygen has
1/10the lifetime in nondeuterated water. Nonetheless, these
by the fullerol in a water suspension.
3.3. Superoxide Production by Fullerol and aqu/nC60
Aggregates. XTT reduction was employed to measure the
production of superoxide in the presence of NADH, a
common reductant found in cells. As shown in Figure 2
dependent manner via type I reaction under medium
pressure (MP) UV light. This indicates that the triplet state
of fullerol is participating in a type I photosensitization
reaction. This is not surprising considering that fullerol
participates in type II singlet oxygen production (Figure 1B
and C) and with the addition of a donor molecule NADH
On the other hand, it was deduced from the TEMP type II
measurements of singlet oxygen (Figure 1A) that aqu/nC60
state as we consider subsequently. Therefore, suspensions
of aqu/nC60would not be expected to participate in type I
photosensitization. This was confirmed by the observation
that aqu/nC60experiments produced less superoxide than
fluences that likely differed by 1 order of magnitude, it is
impossible to make a direct comparison of type I and type
II production rates in this instance.
3.4. TEM and XRD Structural Analysis of Aggregates.
aqu/nC60show that while both suspensions have a similarly
gold-colored appearance, the structure of the aggregates in
consist of nearly spherical aggregates (n-scale) assembled
into larger (m-scale) aggregates of the n-scale spheres. The
diameter of a typical n-scale aggregate as observed by TEM
measurements and may indicate the presence of m-scale
the aqu/nC60has a fractal m-scale structure (45) reflecting
crystalline n-scale aggregates. However, it is the n-scale
the proximity of the majority of C60-C60 contacts, with
implications for the rate of ROS generation due to both the
greater accessibility to the reactive C60surfaces.
Type I and type II photosensitizations by the fullerol and
the lack of these reactions with regard to aqu/nC60 may
indicate a longer lifetime for the triplet state of the hydroxy-
on the aggregates in order to obtain an indication of the
In the fullerol diffractogram (Figure 4 inset), the absence of
a diffraction peak implies a lack of order within the fullerol
aggregate at the n-scale and a greater distance between C60
In contrast, the aqu/nC60diffractogram (Figure 4) shows
a crystalline organization very similar to that of fullerene
fine powder (fullerite). C60molecules in aqu/nC60that has
face centered structure with the same lattice constants as
that of C60fullerite (a ) b ) c ) 14.16 Å). This organization
is apparently retained in spite of the subdivision of particles
of the initial C60powder as they form a stable suspension in
is inherited from the original crystal structure of C60fullerite
before sonication treatment in water. This tighter n-scale
assembly favors triplet–triplet annihilation and reduces
the surface of C60 effectively available for reaction with
approaching oxygen molecules.
3.5. Proposed Framework. We propose a mechanistic
framework (Figure 5) derived from literature and experi-
mental evidence to explain the observed trends in ROS
FIGURE 2. Concentration of superoxide radical as a function of
XTT (100 µM) absorption at suspension concentrations of 10, 20,
and 30 µM. After 5 min of MP UV irradiation fullerol produces
more superoxide in the presence of UV light than NADH (500µM)
alone. Aqu/nC60produces less superoxide than NADH alone.
FIGURE 3. TEM images in fullerol aggregates (left) and aqu/nC60
FIGURE 4. XRD diffractogram of C60and aqu/nC60after suspension
and drying. Inset: XRD diffractogram of fullerol powder.
4178 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 11, 2008
in the structure of aggregates in the aqueous fullerol and
nC60 suspensions. When fullerenes are illuminated under
the appropriate wavelength, the electrons are excited from
the ground state (°C60) to the singlet state (I). In the case of
aqu/nC60and fullerol, this process likely occurs at a higher
rate in aqu/nC60 due to a stronger absorbance at UVA
wavelengths tested in this study. The singlet state (1C60) can
decay in three main manners: fluorescence (II); internal
conversion (III); and intersystem crossing (ISC) (IV). The
former two pathways return the fullerene to the ground-
state while the latter leads to the relaxation of singlet C60to
the triplet state (3C60).
Unaltered C60that is found inside aqu/nC60has a high
promotes fluorescence (II) and internal conversion (III)
(48, 49). Interaction of the singlet state with oxygen can also
result in the triplet state (VI, V). Pathway VI results in the
production of singlet oxygen via type II photosensitization
while pathway V only results in the formation of the triplet
than1C60 in solution allowing it to participate in type II
formation of singlet oxygen to a greater extent than does the
(VII) via interaction with the ground state (°C60) and
triplet (3C60). These processes are promoted by close interac-
4 shows aqu/nC60 cages are keeping their tight crystalline
form even after suspension, while the fullerol suspensions
do not exhibit crystal structure (Figure 4 inset). TEMP
measurements of singlet oxygen confirm that the triplet
lifetime is significantly different in aqu/nC60which has no
trace of type II photosensitization when compared with
lines, type I sensitization (X) occurs when the triplet state
comes in contact with a donor molecule (NADH) that has a
more negative reduction potential than (3C60). Superoxide
(XI). Electrons from this radical could also donate to form
other types of free radicals such as organic radicals.
lived triplet state (∼µs) compared with the short lifetime of
by aqu/nC60measured in Figure 1A validates the proposed
mechanism in that aggregates more closely associated with
oxygen. Both the hydroxylated (fullerol) and underivatized
varieties of C60form stable colloidal suspensions in water.
However, aggregation itself does not imply that these
fullerenes are not able to produce reactive oxygen. The
structure (and likely the size) of the aggregate appear to
the tighter and more structured nature of aqu/nC60 may
prevent ROS production. This occurs despite the higher
absorption of UV light at the frequencies exposed and the
predicted higher reactivity of the unaltered C60cages (52).
Accessibility of water to the fullerene surface may also be
taken in the context of the proposed mechanism and ROS
measurements, indicate that fullerol has a long-lived triplet
clear ramifications for the type I and type II photosensitized
production of ROS in carbon based nanomaterials and thus
should be considered more carefully in future analyses of
these materials for toxicity and application in the aqueous
EPR assistance was provided by Dr. Marian Fabian at Rice
University in Houston, TX. Advice on superoxide detection
via XTT was received from Dr. Fridovich at Duke University.
Funding support for this work was received from National
Science Foundation, US Environmental Protection Agency,
and Centre National de la Recherche Scientifique (France).
Supporting Information Available
Calculation and analysis of projected surface area and
theoretical KOW values of probes used in this study. This
material is available free of charge via the Internet at http://
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