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Highly Selective and Sensitive Photoinduced Electron Transfer

Published on 24 May 2016. Downloaded by University College London on 25/05/2016 17:37:35.

(PET) Based HOCl Fluorescent Probe in Water and Its Endogenous

Received 00th January 20xx,
Accepted 00th January 20xx
Imaging in Living Cells

ChemComm Accepted Manuscript

DOI: 10.1039/x0xx00000x Lijuan Liang,a Chang Liu,a,b Xiaojie Jiao,b Liancheng Zhaoa and Xianshun Zengb, *
www.rsc.org/

A probe based on a phenothiazine-acridine orange conjugate (Ptz- physiological and pathological processes of HOCl at
AO) has been designed and synthesized for sensitive and selective cellular/subcellular level due to its excellent temporal and
detection of HOCl. Ptz-AO has excellent properties, including pH- spatial resolution capability in living cells.6 So far various
independence of fluorescence, high resistance to photobleaching, fluorescent probes with different responsive moieties toward
and response in real time. The value of Ptz-AO was confirmed by HOCl have been designed and developed by taking advantage
exogenous, endogenous and real-time imaging of HOCl in vitro of the strong oxidization ability of HOCl.7 Although many of
using a fluorescence microscope. these probes have been successfully utilized to image HOCl in
cellular environments, few of them could be applied to real-
The reactive oxygen species (ROS) have played important roles time detect HOCl at cellular levels. On the other hand, some of
in cell signaling and homeostasis, such as antiinflammation them suffer from low quantum yield, autoxidation, photo-
regulation, pathogen response and so on.1 Among ROS, bleaching, poor water solubility or lack selectivity toward
hypochlorous acid (HOCl) produced by myeloperoxidase HOCl over other ROS. As a result, it is challenging and highly
(MPO) or induced by a number of soluble stimuli has received desired to prepare novel real-time responsive probes7s for
special attention for its pivotal antimicrobial nature in the investigating the distribution and functions of HOCl at cellular
immune system, which helps inhibit inflammation and regulate level.
cellular fate.2 In nature, HOCl is a weak acid (pKa = 7.63) and It is know that the N-alkylated derivatives of acridine orange
is highly reactive and short-lived in physiological (AO) fluorophore (Chart 1) have several interesting inherent
environments.3 However, keeping a reasonable HOCl photophysical natures which are highly expected for its use as
concentration within the physiological environments is tracers for fluorescence staining caused by the “push-pull”
essentially required for numerous cellular functions. Excessive properties of its internal charge transfer (ICT) excited state,
or uncontrolled production of HOCl can lead to tissue damage arising from the charged electron-withdrawing nitrogen atom
and diseases, including cardiovascular diseases, lung injury
atherosclerosis, osteoarthritis, and cancer.4 Thus, it is urgently Me2N Me2N

required for the development of highly sensitive and selective

methods for further investigation of the complex contributions R N+ S N N+
BF4-
of HOCl to human health. X-

In recent years, a number of analytical techniques, such as

Me2N Me2N
colorimetric, fluorometric, electrochemical, chromatographic,
and optical imaging methods,5 have been proposed for the N-alkylated AO Ptz-AO

detection of HOCl under different conditions. Of all these Chart 1 Structures of N-alkylated acridine orange (AO) and probe Ptz-AO.
analytical techniques, fluorescence imaging shows some sort of Me 2N
uniqueness and irreplaceability for the investigation of the Br

a) b)
S NH S N N+
N c) BF 4-
a.
School of Materials Science and Engineering, Harbin Institute of Technology,
Harbin 150001, China. S
b. Me 2N
Tianjin Key Laboratory for Photoelectric Materials and Devices, Tianjin University
of Technology, Tianjin 300384, China. Fax: (+86)22-60215226; Tel: (+86)22- Ptz 1 Ptz-AO
60216748; E-mail: [email protected].
Electronic Supplementary Information (ESI) available: [details of any Scheme 1 Synthesis of the probe Ptz-AO. Reagents and conditions: a) NaH,
supplementary information available should be included here]. See 1,3-dibromopropane, DMF, r. t., 4 h; b) acridine orange, KI, toluene, reflux,
DOI: 10.1039/x0xx00000x 24 h; c) KBF4, CH2Cl2/EtOH (1:1, v/v), reflux, 3 h.

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COMMUNICATION Journal Name
Me2N Me 2N M e2N
PET PET
PET
HClO
S N N+ O S N N+ + S +N N+
BF4- BF 4- BF4 -

Me2N Me 2N M e2N

Ptz- AO OPtz-AO Ptz- AO cation

Scheme 2 Proposed sensing mechanism of the probe Ptz-AO towards HOCl.

within the chromophore and the two electron-donating amino

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Fig. 1 Fluorescent titration spectra of Ptz-AO (5 μM) in the presence of

moieties. The merits for its use in biological applications are different concentrations of NaClO in H2O; Inset: the fluorescence at 540 nm
that the intense absorption band at ~490 nm meets perfectly the of Ptz-AO (5 μM) as a function of the NaClO concentration. λex = 475 nm,
discrete excitation of the argon laser at 488 nm and emits at ~ Slit = 5 nm, 5 nm.

ChemComm Accepted Manuscript

540 nm while also possessing high quantum yields and good
photostability in varieties of organic and aqueous media. It has
been widely used in cell staining of DNA in apoptosis studies.8
Herein, we report a photoinduced electron transfer (PET) based
HOCl fluorescent probe Ptz-AO, a propylene tethered
conjugate of phenothiazine (Ptz) and acridine orange (AO) dye
(Chart 1). The probe shows high selectivity, high sensitivity
(detection limit of 2.7 nM) and fast response (within 5 seconds)
towards HOCl in water. The marvellous sensing properties of
the probe Ptz-AO enable its use in living cells for the real-time Fig. 2 Change ratio (F - F0)/F0 of fluorescence intensity of Ptz-AO (5 μM)
upon the addition of 2.0 equiv. other ions and anions in H2O; histogram
monitoring of HOCl in INS-1 β-islet cells and RAW264.7
representing the fluorescence enhancement of the probe in the presence
macrophage cells. of other ions and ROS. 1: O21; 2: AcO-; 3: Al3+; 4: Ca2+; 5: Cl-; 6: Cu2+; 7: Fe2+; 8:
The synthetic processes of the target probe were shown in Fe3+; 9: H2O 2; 10: ClO-; 11: Hg2+; 12: K+; 13: Na+; 14: NO2-; 15: ·OH; 16:
.
Scheme 1. The compound 1 was prepared in 65% yield by the ONOO- ; 17: TBHP; 18: Zn2+; 19: NO; 20: O2.-. λex = 475 nm, Slit = 5 nm, 5
reaction of phenothiazine with 1,3-dibromopropane in sodium nm.
hydride (60% dispersing in mineral oil) in dry DMF at room
temperature. Then, Ptz-AO was facilely obtained in 58% yield calculated as 8.95 µM (R = 0.9988) (Fig. S2, ESI†).9 From the
by the reaction of acridine orange with 1 in the presence changes in ClO--dependent fluorescence intensity, the detection
catalytic amount of KI. The anion exchange was achieved by limit of Ptz-AO was estimated to be 2.7 nM (Fig. S3, ESI†).9b
refluxing the CH2Cl2/EtOH (1:1, v/v) solutions Ptz-AO with To explore the selectivity of the probe towards HOCl among
excess KBF4 . The chemical structure of Ptz-AO was confirmed other reactive oxygen species ROS, we simultaneously
by 1H NMR, 13 C NMR, and HRMS spectra. evaluated the response of Ptz-AO to other ions and ROS in
The emission properties of the probe Ptz-AO and those in the water. As shown in Fig. 2, the addition of 2 equiv. of other
. .-
presence of an incremental amount of HOCl were initially ROS, such as O21, H2O2, ONOO- , NO, O2 , and t-BuOOH, and
elucidated in H2O (Fig. 1). The probe showed weak other ions, such as AcO , Cl , NO3 , NO2-, Al3+, Ca2+, Cu2+,
- - -

fluorescence emission (Φ = 0.011) at 540 nm in water due to Fe2+, Fe3+, K+, Na+, and Zn2+ had no obvious effect on the
the strong electron donating property of Ptz moiety which fluorescence emission, only ·OH responded with a slight
quenched the fluorescence emission of AO via the photo- increase (ca. 2.0-fold enhancement) in the fluorescent intensity.
induced electron transfer (PET) process. As can be seen from In contrast, the addition of NaClO resulted in a significant
Fig. 1, the fluorescence titration of the probe with NaClO led to enhancement (over 26-fold) of the fluorescence intensity
a rapid increase of the fluorescence emission intensity at 540 positioned at 540 nm. Thus, Ptz-AO can function as highly
nm due to the HOCl-promoted oxidation of the electron donat- selective fluorescence probe for the HOCl.
ing Ptz moiety to Ptz sulfoxide (OPtz-AO) and Ptz cation (Ptz- To further explore the utility of the probe as a fluorescent
AO cation)10 which prevented the PET process from Ptz to AO chemosensor for HOCl, the competition experiments were
(Scheme 2). High-resolution mass spectra (HRMS) of Ptz-AO, subsequently conducted in which Ptz-AO (5 µM) was first
and the UV-Vis spectra and HRMS of a model compound N- mixed with 2 equiv. of other ions and ROS, and then 2 equiv. of
ethylphenothiazine provided reliable evidence for the oxidation NaClO was added. As shown in Fig. S4, no strong interference
of the probe (Fig. S1a-1c, ESI†). When the ratio of was observed in the presence of other ions and ROS. The
[NaClO]total/[Ptz-AO] reached 5 : 1, higher [NaClO]total did not excellent selectivity towards HOCl among other ions and ROS
lead to further emission enhancement as shown in the inset of suggested that Ptz-AO had the potential to detect HOCl in
Fig. 1. Over a 75-fold enhancement of fluorescence intensity (Φ complex biological environment.
= 0.823) was observed under saturated conditions. The increase At the same time, fluorescence changes of Ptz-AO as a
of the fluorescence emission at 540 nm followed the sigmoidal function of pH were measured in the presence and absence of
curves and the fluorescence turn-on constant (Kturn-on) was NaClO in H2O. The probe showed weak fluorescence signal

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over a pH range of 1-12 (Fig. S5, ESI†). However, it showed HOCl, macrophages RAW 264.7 were successively pre-treated
very strong fluorescence emission upon the addition of 4.0 with bacterial cell wall lipopolysaccharide (LPS) and phorbol
equiv. NaClO from pH 5.0 to pH 11.0. Thus, the results
indicated that the probe Ptz-AO was capable of detecting
hypochlorite and hypochlorous acid in the pH range 5-11
3
because the pKa of HOCl is 7.6.
Finally, the time-dependent fluorescence response and the
photostability of the probe under excitation at 475 nm were
measured in the absence and presence of NaClO. As shown in
Error! Reference source not found., a weak fluorescence
Published on 24 May 2016. Downloaded by University College London on 25/05/2016 17:37:35.

signal was observed with the probe alone. Upon the addition of
NaClO (0.75 equiv.), however, the fluorescence intensity
increased immediately and reached the maximum within 5 s.

ChemComm Accepted Manuscript

Then the fluorescence intensity was stable under continuous Fig. 3 Fluorescence image of INS-1 β-islet cells incubated with the probe Ptz-AO
wavelength 475 nm laser excitation from 5 s to 300 s. The (3 µM). a) fluorescence and brightfield image after staining for 20 min; b)
results suggested that the probe was photostable. Meanwhile, fluorescence and brightfield image after incubated with ClO- (6 µM) for 20 min.
the fast response to ClO - allowed it to be applied to real-time
detecting HOCl in water phase.
In light of the above desirable fluorescence properties of Ptz-
AO as fluorescence probe for the detection of intracellular
HOCl, it was then applied to detect exogenous HOCl in INS-1
cells, a murine β-islet cell line (Fig. ). As shown in Fig. a, when
the INS-1 cells were incubated with the probe Ptz-AO (3 µM)
for 20 min in the growth medium and then washed with DPBS,
a weak fluorescence signal of the probe was present in the cells
with an excitation of green light (488 nm). After treatment with
NaClO (6 µM) for 20 min, however, intense fluorescence signal
of the probe was emerged within the cells (Fig. b). By
comparison with the brightfield image, we found that the
fluorescence signals were only located in the intracellular area,
demonstrating that the probe Ptz-AO was able to detect
exogenous HOCl in cells.
Due to the highly reactive and short-lived properties of Fig. 4 Time-dependent fluorescence image of INS-1 β-islet cells incubated with
HOCl in physiological environments, we then investigated the the probe Ptz-AO (1 µM). Fluorescence (A1) and brightfield (A2) image after
staining for 20 min. Fluorescence (B1-F1) and brightfield (B2-F2) image after
time-dependent fluorescence response of the probe in living
incubated with ClO- (2 µM) for 2, 3, 4, 5, 15 min.
cells to elucidate the real-time responsive properties of the
probe. When INS-1 β-islet cells were incubated with probe (1
µM) for 20 min in PBS at 37 o C, no notable fluorescence
responses were observed (Fig. A). Upon the addition of NaClO
(2 µM), an obvious fluorescence signal was observed within 2
min. Meanwhile, a significant increase of the fluorescence
signals was observed with the incubated time (Fig. B-F). The
integrated optical densities (IOD) of the imagings with the
incubated time indicated that there was a rapid increase of the
fluorescence signals from 2 min to 5 min after the addition of
NaClO (Fig. S7, ESI†). The time-dependent fluorescence
response of the probe (1 µM) in RAW 264.7 cells also showed a
very fast response. There was a marked increase of the
fluorescence signals after the addition of NaClO within 2 min
(Fig. S8, ESI†). Therefore, the probe Ptz-AO was very effective
for HOCl detection and should be suitable for practical
application in vitro.
Finally, we investigated whether the probe Ptz-AO was
Fig. 5 Fluorescence image of RAW264.7 cells incubated with the probe Ptz-
capable of detecting intracellular endogenous HOCl produced
AO (1 µM). a) fluorescence and brightfield image after staining for 20 min;
by resting tissue macrophages under mimic inflammatory b) fluorescence and brightfield image after stimulated by LPS and PMA for
conditions. To elicit the elevated production of endogenous 20 min; c) fluorescence and brightfield image after stimulated by NAC for
30 min, and then stimulated by LPS and PMA for 20 min.

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12-myristate 13-acetate (PMA).11 With LPS/ PMA Tano, Y. Chuman, E. Sakuda, T. Taketsugu, K. Sakaguchi, N.
stimulation, prominent enhanced fluorescence signals were Kitamura and K. Tanino, Chem. Sci., 2015, 6, 1083.
7 (a) Q. A. Best, N. Sattenapally, D. J. Dyer, C. N. Scott and
observed from the living RAW 264.7 cells compared with the M. E. Mc Carroll, J. Am. Chem. Soc., 2013, 135, 13365; (b) J.
controls (Fig. a and 5b), indicating the activation of the probe T. Hou, K. Li, J. Yang, K. K. Yu, Y. X. Liao, Y. Z. Ran, Y.
Ptz-AO under mimic inflammatory conditions. When N- H. Liu, X. D. Zhou and X. Q. Yu, Chem. Commun., 2015, 51,
acetylcysteine (NAC), a general antioxidant,12 was used to treat 6781; (c) H. D. Xiao, K. Xin, H. F. Dou, G. Yin, Y. W. Quan
the cells together with LPS/PMA elicitation, no obvious and R. Y. Wang, Chem. Commun., 2015, 51, 1442; (d) G. H.
Cheng, J. L. Fan, W. Sun, J. F. Cao, C. Hu and X. J. Peng,
fluorescence was observed after incubation with the probe Ptz- Chem. Commun., 2014, 50, 1018; (e) W. Zhang, W. Liu, P.
AO (Fig. c). The result indicated that NAC scavenges Li, J. Q. Kang, J. Y. Wang, H. Wang and B. Tang. Chem.
endogenously generated HOCl from RAW 264.7 cells and Commun., 2015, 51, 10150; (f) B. S. Wang, P. Li, F. B. Yu, P.
Published on 24 May 2016. Downloaded by University College London on 25/05/2016 17:37:35.

effectively inhibited the activation of the probe Ptz-AO. These Song, X. F. Sun, S. Q. Yang, Z. R. Lou and K. L. Han, Chem.
results clearly demonstrated that the probe Ptz-AO can Commun., 2013, 49, 1014; (g) Y. Koide, Y. Urano, K.
Hanaoka, T. Terai and T. Nagano, J. Am. Chem. Soc., 2011,
efficiently detect HOCl produced in stressed cells. 133, 5680; (h) Q. L. Xu, K. A. Lee, S. Y. Lee, K. M. Lee, W.

ChemComm Accepted Manuscript

In conclusion, we have designed and synthesized a novel J. Lee and J. Y. Yoon, J. Am. Chem. Soc., 2013, 135, 9944;
fluorescence probe Ptz-AO by the reaction of N-(3-bromo- (i) J. Zhou, L. H. Li, W. Shi, X. H. Gao, X. H. Li and H. M.
propyl)phenothiazine with acridine orange. Ptz-AO functions as Ma, Chem. Sci., 2015, 6, 4884; (j) J. T. Hou, M. Y. Wu, K.
a highly HOCl-selective fluorescent probe with a fast (within 5 Li, J. Yang, K. K. Yu, Y. M. Xie and X. Q. Yu, Chem.
Commun., 2014, 50, 8640; (k) S. R. Liu and S. P. Wu, Org.
s) and sensitive response, and Ptz-AO exhibits excellent Lett., 2013, 15, 878; (l) J. J. Hu, N. K. Wong, Q. S. Gu, X. Y.
properties as HOCl-specific fluorescent probe for biological Bai, S. Ye and D. Yang, Org. Lett., 2014, 16, 3544; (m) H.
applications, including pH-independence and tolerance to Zhu, J. L. Fan, J. Y. Wang, H. Y. Mu and X. J. Peng, J. Am.
photobleaching during excitation laser irradiation. With the Chem. Soc.,2014, 136, 12820; (n) G. H. Cheng, J. L. Fan, W.
probe Ptz-AO, we successfully conducted exogenous, Sun, K. Sui, X. Jin, J. Y. Wang and X. J. Peng, Analyst, 2013,
138, 6091; (o) J. Liu, Y. Q. Sun, H. X. Zhang, Y. Y. Huo, Y.
endogenous and real-time imaging of HOCl by means of W. Shi and W. Guo, Chem. Sci., 2014, 5, 3183; (p) G. P. Li,
fluorescence microscopy. We anticipate that this probe should D. J. Zhu, Q. Liu, L. Xue and H. Jiang. Org. Lett., 2013, 15,
prove useful for investigation of the wide range of biological 2002; (q) H. Li, F. S. Kim, G. Ren and S. A. Jenekhe, J. Am.
functions of HOCl. Chem. Soc., 2013, 135, 14920.
We gratefully acknowledge the Natural Science Foundation 8 (a) S. Nafisi, A. A. Saboury, N. Keramat, J. F. Neault and H.
A. Tajmir-Riahi, J. Mol. Struct, 2007, 827, 35; (b) S.
of China (NNSFC 21272172), and the Natural Science Moradpourhafshejani, J. H. Hedley, A. O. Haigh, A. R. Pike
Foundation of Tianjin (12JCZDJC21000). and E. M. Tuite, RSC Adv., 2013, 3, 18164.
9 (a) P. Du and S. J. Lippard, Inorg. Chem., 2010, 49, 10753;
Notes and references (b) B. S. Wang, P. Li, F. B. Yu, J. S. Chen, Z. J. Qu and K. L.
Han, Chem. Commun., 2013, 49, 5790.
1 (a) J. D. Lambeth, Free radical Bio. Med., 2007, 43, 332; (b) 10 E. Pelizzetti, D. Meisel, W. A. MuIac, and P. Neta, J. Am.
L. Yuan, L. Wang, B. K. Agrawalla, S. J. Park, H. Zhu, B. Chem. Soc., 1979, 101, 6954.
Sivaraman, J. Peng, Q. H. Xu and Y. T. Chang, J. Am. Chem. 11 S. E. Gomez-Mejiba, Z. Zhai, M. S. Gimenez, M. T. Ashby,
Soc., 2015, 137, 5930; (c) K. P. Kepp, Chem. Rev., 2012, 112, J. Chilakapati, K. Kitchin, R. P. Mason and D. C. Ramirez, J.
5193. Biol. Chem., 2010, 285, 20062.
2 M. Whiteman, P. Rose, J. L. Siau, N. S. Cheung, G. S. Tan, 12 K. C. Sheng, G. A. Pietersz, C. K. Tang, P. A. Ramsland and
B. Halliwell and J. S. Armstrong, Free Radical Biol. Med., V. Apostolopoulos, J. Immunol., 2010, 184, 2863.
2005, 38, 1571.
3 Z. Sun, F. Liu, Y. Chen, P. K. H. Tam and D. Yang, Org.
Lett. 2008, 10, 2171.
4 A. Daugherty, J. L. Dunn, D. L. Rateri and J. W. Heinecke, J.
Clin. Invest., 1994, 94, 437.
5 L. Moberg and B. Karlberg, Anal. Chim. Acta., 2000, 407,
127.
6 (a) X. H. Li, X. H. Gao, W. Shi and H. M. Ma, Chem. Rev.,
2014, 114, 590; (b) X. L. Sun, Q. L. Xu, G. Kim, S. E.
Flower, J. P. Lowe, J. Yoon, J. S. Fossey, X. H. Qian, S. D.
Bull and T. D. James, Chem. Sci., 2014, 5, 3368; (c) T. Guo,
L. Cui, J. N. Shen, R. Wang, W. P. Zhu, Y. F. Xu and X. H.
Qian, Chem. Commun., 2013, 49, 1862; (d) L. Yuan, F. P. Jin,
Z. B. Zeng, C. B. Liu, S. L. Luo and J. S. Wu, Chem. Sci.,
2015, 6, 2360; (e) X. F. Yang, Q. Huang, Y. G. Zhong, Z. Li,
H. Li, M. Lowry, J. O. Escobedo and R. M. Strongin, Chem.
Sci., 2014, 5, 2177; (f) Y. You and W. Nam, Chem. Sci.,
2014, 5, 4123; (g) P. Cheruku, J. H. Huang, H. J. Yen, R. S.
Iyer, K. D. Rector, J. S. Martinez and H. L. Wang, Chem.
Sci., 2015, 6, 1150; (h) J. Pancholi, D. J. Hodson, K. Jobe, G.
A. Rutter, S. M. Goldup and M. Watkinson, Chem. Sci., 2014,
5, 3528; (i) A. Borrmann and J. C. M. Hest, Chem. Sci., 2014,
5, 2123; (j) K. Namba, A. Osawa, A. Nakayama, A. Mera, F.

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