A FRET-based ratiometric two-photon fluorescent probe for dual-channel imaging of nitroxyl in living cells and tissues

Article abstract of DOI:10.1039/c5cc08695g

A FRET-based two-photon fluorescent probe, P-Np-Rhod, which exhibited a fast and high selective ratiometric response to nitroxyl, was first proposed. P-Np-Rhod was successfully applied to two-photon dual-channel imaging of nitroxyl in living cells and tis

Full text of DOI:10.1039/c5cc08695g

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. zhu, M. xiong,
H. Liu, G. Mao, L. Zhou, J. Zhang, X. hu, X. Zhang and W. Tan, Chem. Commun., 2015, DOI:
1
0.1039/C5CC08695G.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Please do not adjust margins
Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/C5CC08695G
Journal Name
COMMUNICATION
A FRET-based ratiometric two-photon fluorescent probe for dual-
channel imaging of nitroxyl in living cells and tissues
Received 00th January 20xx,
Accepted 00th January 20xx
†
a
†a
a
b
a
a
Xiaoyan Zhu , Mengyi Xiong , Hong-wen Liu , Guo-jiang Mao , Liyi Zhou , Jing Zhang , Xiaoxiao
a
a
a
Hu , Xiao-Bing Zhang and Weihong Tan
DOI: 10.1039/x0xx00000x
www.rsc.org/
A FRET-based two-photon fluorescent probe, P-Np-Rhod, which Nevertheless, the biological applications of these fluorescent probes
exhibited a fast and high selective ratiometric response to nitroxyl, are limited by one-photon excitation with short wavelengths, which
was first proposed. P-Np-Rhod was successfully applied to two- could lead to photobleaching of probes, restricted tissue-penetration
photon dual-channel imaging of nitroxyl in living cells and tissues depth, and high cellular autofluorescence. In sharp contrast, two-
with less cross-talk between channels and satisfactory deep-tissue photon microscopy (TPM) approach, which excites a two-photon
imaging depth.
(TP) fluorophore with near-infrared (NIR) laser pulses, can
overcome these shortages and provide improved three-dimensional
Nitroxyl (HNO), the one-electron reduced form of NO, has recently
been found to possess unique biological activity and pharmacology
spatial localization with reduced photodamage to biosamples and
increased tissue penetration depth.12 Despite all these advantages,
only few TP fluorescent turn-on probe has been reported for the
detection of HNO.11k Moreover, this turn-on probe was limited for
1
applications. For example, HNO can inhibit the activity of aldehyde
2
dehydrogenase by interacting with its protein thiols. And it has been
demonstrated to exhibit the potential for treating heart failure
through upregulating the calcitonin gene-related peptide which can
induce vasorelaxation. Moreover, HNO could also be used to treat
practical applications, due to the fact that the fluorescence intensity
measurement within a single detection window may vary with the
experimental conditions, such as the incident laser power and the
distribution of probes. Ratiometric probes, by measuring the changes
of the intensity ratio at two different emission peaks, can provide a
3
alcoholism, mitigate ischemia-reperfusion injury and functionalize
4
as a potential anti-cancer drug. In this light, the development of a
reliable assay method that can monitor HNO in biosystems is highly
desired.
built-in correction to minimize these interferences, resulting in a
more effective system for imaging living cells and tissues.13 It is
Until recently, several analytical methods including colorimetric
approach,5 electron paramagnetic resonance spectroscopy, high
performance liquid chromatography, mass spectrometry8 and
worth pointing out that, although several one-photon excited
ratiometric fluorescent probes have been developed for the detection
of HNO11d, e, g, j, to our best knowledge, no ratiometric TP fluorescent
6
7
9
electrochemical analysis have been developed for HNO detection.
probes for detecting HNO has been reported to date.
In despite of appropriate sensitivity, these methods are unsuitable for
detection of HNO in living systems, due to the complicated
pretreatment and potential destruction of biological samples. In
recent years, fluorescent probes have been widely applied for bio-
imaging of various analytes in living cells, tissues and organisms by
virtue of their high sensitivity, fast response time, and little bio-
damage.10 Recently, based on the reaction of HNO with
triarylphosphine, various fluorescent probes were rationally designed
to discriminate HNO from other biological reductants.11
In this work, by combining the advantages of TPM technique with
ratiometric probe, we designed and synthesized, for the first time, a
ratiometric TP fluorescent probe (P-Np-Rhod, Scheme 1) for HNO
based on a FRET strategy. In this FRET system, a TP naphthalene
derivative served as the energy donor and a rhodol fluorophore was
chosen as the energy acceptor which was modified with a
(diphenylphosphino)-benzoate moiety as a recognition unit for HNO.
In the absence of HNO, the rhodol existed in a non-fluorescent
lactone form and the FRET is off. As reported by previous literatures,
HNO can react with 2-(diphenylphosphino)-benzoate moiety to give
the corresponding aza-ylide, which can attack the adjacent ester
linker in an intramolecular manner to release hydroxyl. And then the
closed spirolactone form was converted to a conjugated fluorescent
xanthene form to induce the occurrence of FRET (Scheme S2, ESI†).
The sensing process was confirmed by both NMR spectroscopy and
mass spectrometry (Fig. S9 and Fig. S10, ESI†). The detailed
synthesis procedures and characterization of all the new compounds
aMolecular Science and Biomedicine Laboratory, State Key Laboratory of
Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering,
Hunan
University,
Changsha
410082,
China.
E-mail:
xbzhang@hnu.edu.cn
；
tan@chem.ufl.edu.
bSchool of Chemistry and Chemical Engineering, Henan
ng, Henan 453007, P. R. China.
Co-first authors.
；
Normal University, Xinxia
†
Electronic Supplementary Information (ESI) available: Synthesis, experimental
details and characterization of new compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
COMMUNICATION
Journal Name
are described in the ESI†.
The fluorescence intensity ratio (I541 nm/I448 nm) of probVeiewP-ANrtipcl-eROhnloinde
DOI: 10.1039/C5CC08695G
15 μM) versus AS concentration after incubation of the probe with
(
AS for 30 min. λex = 370 nm.
The time-dependent fluorescence response of probe P-Np-Rhod
to different concentrations of AS (5, 35, 55 or 100 μM) were
examined by monitoring the change of fluorescent intensity ratio
between the two wavelengths (I541 nm/I448 nm) after the addition of AS
in PBS buffer. As shown in Fig. S4 (ESI†), the fluorescent intensity
ratio (I541 nm/I448 nm) obviously increased and reached a plateau at
around 25 min after the addition of AS, demonstrated that the probe
P-Np-Rhod can provide a rapid response for HNO.
Scheme 1 Chemical structure and the response mechanism of probe
P-Np-Rhodol to HNO.
The spectroscopic properties of the probe P-Np-Rhod were firstly
investigated in buffered (10 mM PBS, pH = 7.4) aqueous
A highly selective response to the target molecule over other
potentially competing molecules is of great significance for a
bioimaging probe with potential application in the complex
biological system. Therefore, the probe P-Np-Rhod was treated
with various biologically relevant analytes to evaluate its selectivity,
including reactive oxygen species, reactive nitrogen species,
reducing agents, small molecule thiols, and biologically related
metal ions. As shown in Fig. 2, no obvious changes of the
fluorescence intensity ratio (I541 nm/I448 nm) were observed in the
presence of these interference species. In contrast, the introduction
of AS could trigger remarkable change of the fluorescence intensity
ratio at I541 nm/I448 nm. This result indicated that the probe P-Np-Rhod
shows a high selectivity for HNO and possesses the ability for
detecting HNO in complex biological samples.
CH
3
CH
2
OH solution (H
2
O/CH
3
CH
2
OH = 1:1, v/v). In the absence of
cyan-colored
HNO, the free probe P-Np-Rhod displayed
a
fluorescence with an emission peak at 448 nm (Fig. 1). After
treatment with enhanced concentrations of AS (from 0 to 100 μM,
Angeli’s salt (AS) acted as the source of HNO in our experiments), a
new fluorescence emission peak at 541 nm appeared and increased
gradually, while the donor’s characteristic emission peak (448 nm)
showed a decreased tendency (Fig. 1), allowing a ratiometric
fluorescence response for the detection of HNO. It is noteworthy that
the large wavelength difference (93 nm) between the two emissions
leads to two well-resolved emission bands for the probe, which is
favourable for dual-channel imaging of HNO in biological samples
with less cross-talk observed. We proceeded to investigate the UV-
absorption properties of the probe. Upon addition of AS, no obvious
absorption changes of the donor occurred, while a new absorption
peak (at 511 nm) belonging to the conjugated xanthene form of the
acceptor increased with an AS dose-dependent pattern. Meanwhile,
the solution changed from colorless to orange, allowing colorimetric
detection of HNO by naked eyes (Fig. S1, ESI†). The remarkable
changes in absorption and fluorescence spectra might be attributed to
the HNO-induced occurrence of FRET between the donor
naphthalene and acceptor rhodol. The Fꢀrster energy transfer
efficiency between the donor and acceptor rhodol was calculated to
be 88.6% (Fig. S2, ESI†). The fluorescence intensity ratio value
between the two wavelengths (I541 nm/I448 nm) varied from 0.13 to 4.67
with the concentration of HNO changed from 0 to 100 μM (Fig. 1).
The detection limit for HNO was calculated to be 5.9×10-7
3σ/slope, Fig. S3, ESI†), indicating that the probe P-Np-Rhod
exhibited a high sensitivity for HNO.
M
(
Fig. 2 Fluorescence response of the probe P-Np−Rhod (15 μM)
toward various analytes in PBS buffered (10 mM, pH = 7.4) aqueous
EtOH solution (1:1, v/v) at room temperature for 30 min. The
fluorescence intensity ratio (I541 nm/I448 nm) was plotted versus
substances: 1. Probe only (15 μM); 2-12. Ca , Ascorbic Acid, NO
2
-,
2
+
-
2-
+
2+
-
+
2+
2+
3+
N
1
t
3
, S , K , Zn , NO
3
, Na , Fe , Mg (1 mM); 13. Fe (100 μM),
-
-, •OH, TBHP,
4-15. Cys, GSH (3 mM); 16-23. H
2
O
2
, ClO , NO, O
2
-
•
OBu, ONOO (500 μM); 24. AS (100 μM). λex = 370 nm.
The influence of pH on the fluorescence response of P-Np-
Rhod to HNO was further investigated. As shown in Fig. S5
ESI†), in the absence of HNO, the probe P-Np-Rhod was pH
(
insensitive over a pH range of 5.0-10.0, indicating that the ester
group was stable in this pH range. Upon addition of AS, the
maximal fluorescence intensity ratio (I541 nm/I448 nm) appeared in
Fig. 1 Fluorescence emission spectra of probe P-Np-Rhod (15 μM) the pH range of 6.0-7.8, which demonstrated that probe P-Np-
in the presence of various concentrations of AS (0-100 μM) in PBS Rhod can be employed to detect HNO in physiological systems.
buffered (10 mM, pH = 7.4) aqueous EtOH solution (1:1, v/v). Inset: With the pH value larger than 8.0, fluorescence intensity ratio
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 4
ChemComm
Journal Name
COMMUNICATION
(
I
541 nm/I448 nm) showed a decreased tendency due to the
To further investigate the applications of P-VNiepw-ARrtihcleoOdnliinne
decreased decomposition rate of AS into nitroxyl under the fluorescence imaging of living samplDeOs,I: 1T0P.103e9x/Cci5tCeCd08d6u9a5lG-
strong basic conditions.14
channel imaging of P-Np-Rhod for HNO in rat liver tissue
The TP active cross-section (δΦ) value of P-Np-Rhod and Np- slices was then performed (Fig. 5). The changes of fluorescence
Rhod were respectively estimated to be 48 GM and 27 GM at 740 signal with scan depth were recorded by TPM in the Z-scan
nm (Fig. S6, ESI†), indicating high capability of the probe P-Np- mode. In the absence of AS, the tissue slices incubated with
Rhod for detecting HNO with TP mode. In the absence of HNO, P- only P-Np-Rhod exhibited obvious fluorescence at the cyan
Np-Rhod showed a fluorescence emission peak at 448 nm under channel with an imaging depths of 40-150 μm. In contrast,
excitation with 740 nm femtosecond pulses. However, a new when the tissue slices were incubated with P-Np-Rhod (50
fluorescence emission peak at 541 nm appeared while the donor’s μM
emission peak at 448 nm vanished in the presence of HNO (Fig. 3). 60 min, relatively significant fluorescence was observed at the
The probe P-Np-Rhod was demonstrated to possess potentail ability yellow channel with an imaging depths of 20-130 μm
) for 60 min, and then treated with 1 mM AS for another
,
for ratiometric TP fluorescent imaging of HNO.
followed with a remarkable decrease at the cyan channel. These
results indicated that P-Np-Rhod possesses excellent tissue
penetration and staining ability as well as ratiometric dual-
colour (cyan and yellow) imaging performance with less cross-
talk between dual emission channels.
Fig. 3 TP excited emission spectra of P-Np-Rhod (black line) and
Np-Rhod (red line). TP excitation at 740 nm in PBS/EtOH (1:1, v/v,
1
0 mM, pH=7.4).
Encouraged by the remarkable features of the probe P-Np-
Fig. 4 TP fluorescence images of HNO in live HeLa cells with P-
Np-Rhod. (a) TP fluorescence images HeLa cells incubated with
only probe P-Np-Rhod (15 μM) for 30 min in the cyan channel; (b)
images of (a) in the yellow channel; (c) Merged images of (a) and
Rhod including high selectivity and sensitivity, rapid response
and good TP fluorescence properties, we next investigated
whether this probe can be applied in TP excited dual-channel
imaging of HNO in living cells. MTT assays were first
performed to evaluate the potential cytotoxicity of P-Np-Rhod
and Np-Rhod at various concentrations against HeLa cells. The
results indicate that both P-Np-Rhod and Np-Rhod have no
obvious cytotoxicity to live cells under the experimental
conditions. (Fig. S7, ESI†). We then examined the applicability
of the probe for detecting HNO in living cells, with results
shown in Fig. 4. The HeLa cells incubated with only the probe
P-Np-Rhod (15 μM) displayed a strong TP fluorescence signal
in the cyan channel but weak fluorescence signal in the yellow
channel, corresponding to strong fluorescence of the donor and
weak fluorescence of the acceptor due to its spirolactone form.
In contrast, HeLa cells incubated with P-Np-Rhod (15 μM) for
(
b); (d, g) TP fluorescence images of HeLa cells incubated with P-
Np-Rhod (15 μM) for 30 min, then with AS (100 μM or 200 μM)
for 30 min in the cyan channel; (e, h) TP fluorescence images of (d,
g) in the yellow channel; (f) Merged images of (d) and (e); (i)
Merged images of (g) and (h). λex = 740 nm, cyan channel: λem
70-530 nm; yellow channel 550-650 nm. Scale bar: 50 μm.
=
4
3
0 min, and then treated with AS (100 μM or 200 μM)
exhibited a significant fluorescence enhancement in the yellow
channel and concomitant decrease in cyan channel,
a
corresponding to strong fluorescence from the open-ring
acceptor and the weak fluorescence from the donor due to the
effective FRET process triggered by HNO. All above results
showed that P-Np-Rhod was cell membrane permeable and Fig. 5 TP fluorescence imaging of a rat liver frozen slice stained
could be successfully applied to TP-excited ratiometric dual- with P-Np-Rhod (50 μM) at ∼90 μm for 60 min (a, b), followed by
channel imaging of HNO in living cells.
treatment with AS (1 mM) and incubated for another 60 min (c, d).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
COMMUNICATION
Journal Name
The images were collected at 450-530 nm (cyan channel, a, c) and 9. (a) S. A. Suarez, D. E. Bikiel, D. E. Wetzler, M. A.VieMwaArrttiicleanOdnlinFe.
Doctorovich, Anal. Chem., 2013, 85, 10D26O2I:;1(0b.1)0S3.9A/C. 5SCuCa0re8z6,9M5G.
H. Fonticelli, A. A. Rubert, E. de la Llave, D. Scherlis, R. C.
Salvarezza, M. A. Marti and F. Doctorovich, Inorg. Chem., 2010,
5
40-650 nm (yellow channel, b, d) upon excitation at 740 nm with
femtosecond pulses. Scale bars: 200 μm
4
9, 6955.
0. (a) J. Fan, M. Hu, P. Zhan and X. Peng, Chem. Soc. Rev., 2013, 42,
9; (b) J.-T. Hou, M.-Y. Wu, K. Li, J. Yang, K.-K. Yu, Y.-M. Xie and
In summary, we have developed a ratiometric TP fluorescent
probe P-Np-Rhod for detecting HNO for the first time. The
diphenylphosphinobenzoyl group was employed as a recognition
unit and a FRET cassette, termed Np-Rhod, acted as the fluorescent
reporter. This ratiometric probe P-Np-Rhod can rapidly measure
1
2
X.-Q. Yu, Chem. Commun., 2014, 50, 8640; (c) X. Li, X. Gao, W.
Shi and H. Ma, Chem. Rev., 2014, 114, 590; (d) L. Yuan, W. Lin, K.
Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622.
HNO with large emission shift (93 nm), high sensitivity and high 11. (a) K. N. Bobba, Y. Zhou, L. E. Guo, T. N. Zang, J. F. Zhang and S.
selectivity. More importantly, P-Np-Rhod could be succesfully
applied for TP fluorescence imaging of HNO in living cells and
tissues with less cross-talk between dual emission channels. Far
more than a promising tool to investigate the biological functions of
HNO, this FRET-based TP strategy might also be used to design
dual-channel imaging probes for other biological researches.
This work was supported by the National Key Scientific Program
of China (2011CB911000), the National Key Basic Research
Program of China (2013CB932702), NSFC (Grants 21325520,
Bhuniya, RSC Adv., 2015, 5, 84543; (b) X. Jing, F. Yu and L. Chen,
Chem. Commun., 2014, 50, 14253; (c) K. Kawai, N. Ieda, K.
Aizawa, T. Suzuki, N. Miyata and H. Nakagawa, J. Am. Chem. Soc.,
2
013, 135, 12690; (d) C. Liu, Z. Cao, Z. Wang, P. Jia, J. Liu, Z.
Wang, B. Han, X. Huang, X. Li, B. Zhu and X. Zhang, Sens.
Actuators, B, 2015, 220, 727; (e) C. Liu, H. Wu, Z. Wang, C. Shao,
B. Zhu and X. Zhang, Chem. Commun., 2014, 50, 6013; (f) P. Liu,
X. Jing, F. Yu, C. Lv and L. Chen, Analyst, 2015, 140, 4576; (g) H.-
M. Lv, Y. Chen, J. Lei, C.-T. Au and S.-F. Yin, Anal. Methods, 2015,
7, 3883; (h) G.-J. Mao, X.-B. Zhang, X.-L. Shi, H.-W. Liu, Y.-X. Wu,
2
1327009, J1210040, 21177036), the Foundation for Innovative
L.-Y. Zhou, W. Tan and R.-Q. Yu, Chem. Commun., 2014, 50,
5790; (i) Z. Miao, J. A. Reisz, S. M. Mitroka, J. Pan, M. Xian and S.
B. King, Bioorg. Med. Chem. Lett., 2015, 25, 16; (j) H. Zhang, R.
Liu, Y. Tan, W. H. Xie, H. Lei, H. Y. Cheung and H. Sun, ACS Appl.
Mater. Interfaces, 2015, 7, 5438; (k) K. Zheng, W. Lin, D. Cheng,
H. Chen, Y. Liu and K. Liu, Chem. Commun., 2015, 51, 5754.
2. (a) D. Kim, H. G. Ryu and K. H. Ahn, Org. Biomol. Chem., 2014,
Research Groups of NSFC (Grant 21221003), National
Instrumentation Program (2011YQ030124). National Natural
Science Foundation of China (21505032). Key scientific research
project of higher education of the Henan province (15A150016)
1
1
8
2, 4550; (b) H. M. Kim and B. R. Cho, Acc. Chem. Res., 2009, 42,
63; (c) H. M. Kim and B. R. Cho, Chem. Rev., 2015, 115, 5014.
notes and references
1
. (a) F. Doctorovich, D. Bikiel, J. Pellegrino, S. A. Suárez, A. Larsen
and M. A. Martí, Coord. Chem. Rev., 2011, 255, 2764; (b) J. C.
Irvine, R. H. Ritchie, J. L. Favaloro, K. L. Andrews, R. E. Widdop
and B. K. Kemp-Harper, Trends Pharmacol. Sci., 2008, 29, 601; (c)
K. M. Miranda, Coord. Chem. Rev., 2005, 249, 433.
1
3. M. H. Lee, J. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, 44,
185.
4. A. S. Dutton, J. M. Fukuto and K. N. Houk, J. Am. Chem. Soc.,
004, 126, 3795.
4
1
2
2. E. G. DeMaster, B. Redfern and H. T. Nagasawa, Biochem.
pharmacol., 1998, 55, 2007.
3
. N. Paolocci, T. Katori, H. C. Champion, M. E. St John, K. M.
Miranda, J. M. Fukuto, D. A. Wink and D. A. Kass, Proc. Natl.
Acad. Sci. U. S. A., 2003, 100, 5537.
4. (a) A. J. Norris, M. R. Sartippour, M. Lu, T. Park, J. Y. Rao, M. I.
Jackson, J. M. Fukuto and M. N. Brooks, Int. J. Cancer, 2008, 122,
1
905; (b) P. Pagliaro, D. Mancardi, R. Rastaldo, C. Penna, D.
Gattullo, K. M. Miranda, M. Feelisch, D. A. Wink, D. A. Kass and
N. Paolocci, Free Radical Biol. Med., 2003, 34, 33; (c) N. Paolocci,
M. I. Jackson, B. E. Lopez, K. Miranda, C. G. Tocchetti, D. A. Wink,
A. J. Hobbs and J. M. Fukuto, Pharmacol. Ther., 2007, 113, 442.
. (a) S. E. Bari, M. A. Marti, V. T. Amorebieta, D. A. Estrin and F.
Doctorovich, J. Am. Chem. Soc., 2003, 125, 15272; (b) K. P.
Dobmeier, D. A. Riccio and M. H. Schoenfisch, Anal. Chem., 2008,
5
80, 1247; (c) M. A. Marti, S. E. Bari, D. A. Estrin and F.
Doctorovich, J. Am. Chem. Soc., 2005, 127, 4680.
6
. (a) U. Samuni, Y. Samuni and S. Goldstein, J. Am. Chem. Soc.,
2
010, 132, 8428; (b) Y. Xia, A. Cardounel, A. F. Vanin and J. L.
Zweier, Free Radical Biol. Med., 2000, 29, 793.
7
. S. Donzelli, M. G. Espey, D. D. Thomas, D. Mancardi, C. G.
Tocchetti, L. A. Ridnour, N. Paolocci, S. B. King, K. M. Miranda, G.
Lazzarino, J. M. Fukuto and D. A. Wink, Free Radical Biol. Med.,
2
006, 40, 1056.
8
. M. R. Cline, C. Tu, D. N. Silverman and J. P. Toscano, Free Radical
Biol. Med., 2011, 50, 1274.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins