A fluorescent Arg-Gly-Asp (RGD) peptide-naphthalenediimide (NDI) conjugate for imaging integrin αvβ3in vitro

Article abstract of DOI:10.1039/c4cc08265f

We have developed a fluorescent peptide conjugate (TrpNDIRGDfK) based on the coupling of cyclo(RGDfK) to a new tryptophan-tagged amino acid naphthalenediimide (TrpNDI). Confocal fluorescence microscopy coupled with fluorescence lifetime imaging (FLIM) map

Full text of DOI:10.1039/c4cc08265f

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DOI: 10.1039/C4CC08265F
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ARTICLE TYPE
A Fluorescent Arg–Gly–Asp (RGD) Peptide ꢀ Naphthalenediimide (NDI)
Conjugate for Imaging Integrin αvβ3 In Vitro
Zhiyuan Hu#¶*, Rory L. Arrowsmith¶, James A. Tyson¶, Vincenzo Mirabello¶, Haobo Ge¶, Ian M.
Eggleston§, Stan W. Botchway‡, G. Dan Pantos¶*, Sofia I. Pascu¶*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
considerable attention recently because of excellent electron
50 accepting properties and solubility in biocompatible media as well
as organic solvents 6ꢀ7. NDIs and their derivatives are flat, aromatic
molecules which have fluorescent properties, suitable for singleꢀ
and twoꢀphoton cellular imaging and are easily functionalisable
with amino acids.7 These make NDI derivatives attractive
55 candidates as building blocks for optical imaging probes,
particularly as NDIs have strong absorption and fluorescence
emissions in the visible and nearꢀIR wavelengths. Previous work on
aminoacidꢀfunctionalised NDIs showed that, despite quantum
yields that rather low in aqueous media (less than 0.005 with
60 respect to [Ru(bipy)3PF6]’ likely due to nonꢀradiative decay
pathways emerging from aromatic stacking and internal hydrogen
bonds)8, such materials are taken up by cancer cells in preference
with respect to healthy cells. Here, the first NDIꢀpeptide imaging
probe incorporating a cancer specific targeting peptide (cyclic
65 RGDfK) and an amino acid (LꢀTrp) was designed and synthesised
in two simple synthetic steps through the EDCꢀmediated coupling
to the strongly fluorescent tryptophanꢀNDI, which emits broadly in
both singleꢀ and twoꢀphoton excitation with maxima above 500 nm.
We have developed
(TrpNDIRGDfK) based on the coupling of cyclo(RGDfK) to a
new tryptophanꢀtagged amino acid naphthalenediimide
10 (TrpNDI). Confocal fluorescence microscopy coupled with
fluorescence lifetime imaging (FLIM) mapping, single and
twoꢀphoton fluorescence excitation, lifetime components and
corresponding decay profiles were used as parameters able to
investigate qualitatively the cellular behavior regarding the
15 molecular environment and biolocalisation of TrpNDI and
TrpNDIꢀRGDfK in cancer cells.
a
fluorescent peptide conjugate
Tumor angiogenesis is the process of growing new blood vessels
that can assist cancerous growths through the supply of nutrients
20 and oxygen as well as removing waste products.1 Angiogenesis is
an important feature of the spread of a tumor, given that single
cancer cells can escape into blood vessels and can also be
transported to distant sites,2 thus generating secondary tumors.
Angiogenesis is a potential target for combating cancer as it plays a
25 key role in tumor metastasis and growth. The use of specific
compounds that may inhibit new blood vessels formation at the
tumor site may help to switch off the metastasis, but their intimate
behavior at the cellular level remains a matter of intense
investigations: as such, the αvβ3 integrin peptide is one of the most
30 important molecular markers involved in the mechanism of cell
adhesion to the extracellular matrix (ECM). The integrin is highly
expressed during angiogenesis and plays an important role in
transferring signals from the extraꢀcellular environment to the
intracellular compartment.3 The αvβ3 integrin is absent in resting
35 endothelial cells and most normal organ systems, rendering it a
potential target for antiꢀangiogenic cancer therapy. Tumor
progression and metastasis of breast cancer, glioma, melanoma and
ovarian carcinoma are all linked to αvβ3 integrin overexpression
during tumor angiogenesis. It has been found that several proteins
40 such as vitronectin, fibrinogen and fibronectin can bind to αvβ3
integrin via the same amino acid sequence arginineꢀglycineꢀaspartic
acid, or RGD.4 It has been shown that cyclo(RGDfK) is one of the
most prominent structures for development of molecular imaging
agents for the assessment of αvβ3 expression. This pentapeptide,
45 cyclo(ꢀArgꢀGlyꢀAspꢀDPheꢀValꢀ), was developed by Kessler and
coꢀworkers to show high affinity and selectivity for αvβ3 integrin.5
We, and others, have studied the supramolecular chemistry of
functionalised naphthalene diimides: NDI molecules have attracted
H
N
NH2
OH
O
O
O
N
O
O
N
O
O
O
O
O
O
O
N
O
4 eq. NHS
6 eq. EDC.HCl
HO
O
O
N
O
O
NH
O
HN
O
NH
OH
N
O
O
HN
DMF, r.t., 12 hrs
O
DMF, Et3N
O
N
O
µW, 5 min, 140 o
C
2
1
O
2a
OH
O
O
O
N
H
HN
NH
O
NH HN
O
H
N
O
HN
NH2
NH
NH
H2
N
O
O
NH
O
O
NH
N
N
O
HN
O
N
O
H
OHN
NH
NH
O
HN
OH
HN
H2
N
O
O
H2
N
O
NH
O
N
O
H
NH HN
O
NH
HN
H
N
O
O
3
4
O
HO
70
Scheme 1. Synthesis of tryptophanꢀNDIꢀRGDfK, compound 4
To shed light into the fate of bio/medical imaging probes in a
cellular environment, the uptake of the new compounds 2 and 4
75 shown in Scheme 1 was investigated using Fluorescence Lifetime
Imaging Microscopy (FLIM) coupled with multiphoton confocal
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DOI: 10.1039/C4CC08265F
fluorescence microscopy: this combination of methods, together
with MTT assays already provided reliable means to probe directly
the environmental effects upon uptake and biolocalisation of
aromatic compounds as probes inside living cells.7b, 8ꢀ9
The cycloꢀ(RGDfK) was chosen in particular for the potential to
selectively bind to prostate cancer cells that overexpress the
specific target receptor. Furthermore, the NDIs plus cycloꢀ
(RGDfK) conjugates were used to image cancer cells in vitro.
A series of purposeꢀmade RGDfK peptides were synthesised
The quantum yield of TrpNDI (2) was determined to be 0.002 in
DMSO, with fluorescein in aqueous 0.1 M NaOH as the
reference10. This was in line with that found for related NDIs in
55 aqueous environments and, since earlier studies on NDIs and
related molecules showed that this does hamper the cellular tracing
by multiphoton confocal fluorescence imaging, fluorescence
lifetime imaging microscopy (FLIM) and confocal laser scanning
microscopy were utilised as the imaging tools of choice for the
60 investigations into the probe behavior in living cells. Furthermore,
5
10 on a laboratory scale using a standard Fmoc solid phase synthesis
MTT assays (ESI) showed that at concentrations of ca. 100 ꢁM
protocol, as described in ESI. The resulting cyclic peptide 3 was
both compounds 2 and 4 are biocompatible within 48 h observation
time towards both FEKꢀ4 (a healthy cell line) and PCꢀ3 (prostate
cancer cell line). ESI describes in full the results of the cellular
65 viability assays. The stability of 4 with respect to decomposition
and the retention of corresponding in vitro fluorescence emissions
were probed in living cells for both TrpNDI (2) and TrpNDIꢀ
RGDfK (4). In order to detect TrpNDI (2) and its RGDfK
derivative (4) inside cancer cells, FLIM was used here for mapping
70 the lifetime of the fluorophores’ emission and also to shed light
upon their cellular biodistribution: this was found, in control
experiments (described in ESI) to be significantly removed from
cellular autofluorescence. Interestingly, unlike related NDIs,
compounds 2 and 4 do not localise in the cells’ nuclei but
1
successfully isolated and characterised by H NMR in d6ꢀDMSO
(including NOESY to verify cyclisation), ESI/MS, MALDI and
HPLC. A new compound, tryptophan substituted NDI (TrpNDI,
15 compound 2) was synthesised from LꢀTryptophan and 1,4,5,8ꢀ
naphtalenetetracarboxylic dianhydride (1), using a rapid and
straightforward microwaveꢀassisted method (Scheme 1), in
almost quantitative yield. The molecular structure and
supramolecular selfꢀassembly of TrpNDI (2) was determined in
20 the solid state by single crystal Xꢀray crystallography (Figure 1).
Crystals suitable for analysis were grown from DMSO and
synchrotron single crystal Xꢀray diffraction data was obtained for
2. The molecules are held together in a hydrogen bonded network
formed between the tryptophan NH of one molecule and the
25 carboxylic carbonyl C=O from neighbouring molecules (N1···O2 75 throughout the cytoplasm. Figures 2 and 3 show single photon
2.868 Å, 162.3°; N4···O7 2.862 Å, 168.4°). This supramolecular
structure is further reinforced by πꢀπ donorꢀacceptor interactions
between the tryptophan residue and the NDI core (avg. π···π
distance 3.233Å, with a 5.9° angle between the two avg. π
30 planes). The carboxylic OH is involved in hydrogen bond
interactions with the solvent molecules (DMSO and H2O).
Crystal data and structure refinement parameters are included in
ESI.
confocal laser scanning microscopy (CLSM) images of TrpNDI (2)
and TrpNDIꢀRGDfK (4) in PCꢀ3 cells (λex = 543 nm). TrpNDI (4)
shows emission upon exciting at either 405 nm, 488 nm and 543
nm, but TrpNDIꢀRGDfK only shows emission after exciting at 543
80 nm alone. This strongly suggests that following the tagging of 2
with two c(RGDfK) units, the incorporation of this peptide narrows
the excitation wavelength range necessary to bypass the cellular
autofluorescence and to trace compound 4 in vitro.
85
35
Figure 2. Singleꢀphoton laserꢀscanning confocal microscopy of PCꢀ3
cells incubated for 25 min. at 37 oC with Compound 2 (100 ꢁM in 1: 99 %
DMSO: serum free medium): (aꢀd) (λex = 405 nm, broad observed weak
90 emission between 515ꢀ530 nm); (eꢀh) (λex = 488 nm, broad observed
emission both in the red 605ꢀ675 nm and green 515ꢀ530 nm channels).
Micrographs a and e show the brightꢀfield images, confirming a ‘healthy’
cellular morphology within this experiment time; d and h represent
overlays of the micrographs (aꢀbꢀc) and (eꢀfꢀg) respectively.
Figure 1. Molecular structure of Compound 2: (a) Side and (b) crystal
unit cell packing views of TrpNDI where all hydrogen atoms and a
disordered solvent molecule (DMSO) have been removed for clarity. (c) a
40 view along the a axis rotated by 90° in the b/c plane showing disordered
H2O and DMSO solvating molecules.
With compound 2 in hand, the corresponding NDIꢀRGDfK
conjugate (TrpNDIꢀRGDfK, 4) was synthesised by coupling the
45 EDCꢀfunctionalised TrpNDI intermediate (compound 2a, described
in ESI) to the amino group of lysine residue of the deprotected
cyclic peptide RGDfK (compound 3, as shown in Scheme 1 and
described in ESI). The novel NDIꢀpeptide conjugate (TrpNDIꢀ
RGDfK) was purified and isolated by semi preparativeꢀHPLC on a
50 milligram scale and characterised by MALDI mass spectrometry
(given in ESI).
95 Scalebar: 5 ꢁm.
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Both TrpNDI 2 and TrpNDIꢀRGDfK 4 (10 mM stock solutions
in DMSO, 2P excitation 810 nm) were found to decay as two
40 component systems, with minor components (τ2) in the order of
several nanoseconds and major components (τ1) in the order of
several hundred picoseconds (Table 1). In DMSO, at 100 ꢁM
Compound 2 was also found to decay as a three component system,
with a τ1 of 0.1 ns (50%), with a τ2 of 0.7 ns (22.8%) and a τ3 of 3.2
45 ns (27.2%), the χ2 was 1.18. The TSPC spectrum of TrpNDIꢀ
RGDfK displays a very close decay behavior to that of TrpNDI
(Table 1, Figure 4 and ESI) and in both cases evidence of some
aggregation (in line with the Xꢀray structure observations, Figure 1)
may account for the extremely short lifetime components present.
50
Next, living PCꢀ3 cells were plated on Petri dishes incorporating
a glass cover slip and allowed to adhere for 12 h. Background
lifetime readings were recorded using fluorescence lifetime
imaging microscopy (FLIM) before the addition of compound. The
Petri dishes containing the cells were then mounted on the
Figure 3. Singleꢀphoton laserꢀscanning confocal microscopy (λex = 543
nm, observed emission: 605ꢀ675 nm) of PCꢀ3 cells incubated for 25 mins
at 37 oC with: (aꢀc) Compound 2 (100 ꢁM in 1: 99 % DMSO: serum free
medium); (dꢀf): Compound 4 (100 ꢁM in 1: 99 % DMSO: serum free
medium). Scalebar 5 µm. The biolocalisation of 2 is uniform throughout
the cytoplasm whereas that of 4 occurs mainly in punctuated, vesicular
regions in the cytoplasm.
5
55 microscope stage and kept at 37 °C during imaging, which was
performed immediately.
Table 1. Lifetime decay constants for compounds 2 and 4 point decay
recorded in pure DMSO solutions (10 mM)
10
Two photon imaging experiments were carried out given that
standard dyes coꢀstaining assays were not conclusive in pinpointing
specific organelles for dyes’ biolocalisation. To investigate the
effect of the environment on the new probes, lifetime
measurements were recorded using timeꢀcorrelated single photon
Compounds
τ1 / ns
0.6
0.6
A1
45.9
55.4
%
τ2 / ns
2.8
2.9
A2
54.1
44.6
%
χ2
1.2
1.0
2
4
15 counting with an excitation wavelength of 810 nm and the emission
measured at 360ꢀ580 nm both in solutions and in living cells. The
solution measurements of TrpNDI and TrpNDIꢀRGDfK were
carried out in DMSO solutions with concentration of 10 mM
(stock) and 100 ꢁM. Figure 4 shows an overlay of the twoꢀphoton
20 excitation lifetime decay curves for Compounds TrpNDI 2 and
TrpNDIRGDfK 4 in solution. Lifetime components data were
processed using SPCImage analysis software (Becker and Hickl,
Germany) or Edinburgh Instruments F900 TCSPC analysis
software. The data profile includes the goodness of fit of the decay
25 curves as χ2, and the lifetime of each component and their
weighting of each component are given in Tables 1 and 2. The χ2
value of 1.0 means an optimal single exponential fit of this
measurement. If the χ2 value is more than 1.3 there is an incomplete
single exponential fit of this measurement. High χ2 values (>1.5)
30 mean either significant noise within the TCSPC setup (electronics
and/or excitation source) or more than one component decay
profile.
Compounds 2 and 4 were dissolved in DMSO and added to the
60 cells to achieve a final concentration of dye of 100 ꢁM in EMEM
medium containing 1% DMSO (v/v). Cell uptake was monitored
after 20 min incubation. Furthermore, precursor compound 2 was
also incubated at a final concentration of 500 µM and 5 % DMSO
(v/v) (ESI). Measurements were recorded for the whole field of
65 view. The FLIM images obtained for TrpNDI (2) and its RGDfK
derivative (4) are displayed in Figures 5 and 6, including an
intensity map showing spatial variations in fluorescence
emission, the lifetime map displaying the distribution of different
fluorescence decay lifetimes throughout the cellular cytoplasm
70 and the profiles of the corresponding lifetime distributions. The
excited state lifetime data (Table 2) show that compounds 1, 2
and 4 have two lifetime components: a major component A1 for
(τ1) and a minor component A2 for (τ2). Compound 2 possessed
lifetime components in PCꢀ3 cells closely matched those
75 observed in DMSO solution, with compound 1 displaying a τ1
and τ2 in a cellular environment similar to the τ2 and τ3 found in
DMSO. Interestingly in PCꢀ3 cells both lifetime components
were shorter than observed in solution, indicating less stable
excited state in the cellular environment. Furthermore, a control
80 in PCꢀ3 cells showed entirely different lifetime components from
the case when 2 or 4 were incubated with this cell line (Table 2).
This confirms that the compounds do indeed enter the cells, and
in line with single photon CLSM measurements, both 2 and 4
predominantly localise within the cytoplasm with negligible
85 nuclear uptake, unlike the related iodineꢀfundtionalised Lꢀphenyl
alanine NDI reported earlier.8 Interestingly, although both 2 and 4
are biocompatible with the living cells used hereby within the
imaging experiments’ timescales, they exhibit different
fluorescence lifetime values, but which are comparable with
90 those resulting from the solution experiments and also show
clearly distinct cellular distributions in vitro. This is not
surprising given the presence of the RDG targeting peptide in 4,
molecular size and shape differences and the fact that compound
4 is more likely than 2 to form large lipophilic cations upon
95 protonation.
Figure 4. Twoꢀphoton timeꢀcorrelated single photon counting:
35 fluorescence decay traces and corresponding fitted curves for the lifetime
determinations (λex = 810 nm, TrpNDI 2, TrpNDIRGDfK 4, 10 mM, pure
DMSO, 5.7 mW).
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DOI: 10.1039/C4CC08265F
250
200
150
100
50
0
2
4
Lifetime (ns)
6
8
a
b
c
Figure 5. Twoꢀphoton laser confocal fluorescence with λex = 810 nm. (a)
Typical micrograph of PCꢀ3 cells incubated for 20 min at 37 oC with
compound 2 (100 ꢁM in 1: 99 % DMSO: EMEM showing compound 2
located throughout in cytoplasm, lifetime mapping; (b) 2ꢀphoton
fluorescence emission intensity image and (c) corresponding average τ
lifetime distribution curve and lifetime scaleꢀbar.
10 Figure 6. Twoꢀphoton laser confocal fluorescence with λex = 810 nm.
Typical micrograph of PCꢀ3 cells incubated for 20 min at 37 oC with
compound 4 (100 ꢁM in 1: 99 % DMSO: EMEM showing compound 4
mainly located in vesicular regions in the cytoplasm: lifetime mapping
(a), intensity image (b), and corresponding average τ lifetime distribution
15 curve and lifetime scaleꢀbar (c).
5
Table 2. Typical lifetime decay constants for compounds 2 and 4 evaluated in cellular regions showing uptake in
PCꢀ3 cancer cells from Figures 5 and 6, as well as Control (ESI). Individual lifetime components and their contributions are given.
Compound
τ1 / ns
FHHM / ns
A1
%
τ2 / ns
FHHM / ns
A2
%
χ2
2
4
1.0
0.3
1.3
0.5
0.1
0.4
80.1
75.7
79.8
4.0
1.9
5.2
2.3
0.73
2.3
20.1
24.3
20.2
1.16
1.00
1.29
Control
† Electronic Supplementary Information (ESI) available. [Xray data for
TrpNDI, analytical and semiꢀpreparative HPLC data, Synthesis of the
60 RGDfK peptide, Cell culture and control experiments, MTT assays, ESIꢀ
MS and MALDI spectra and HPLC are included in supplementary
materials.]. See DOI: 10.1039/b000000x/
In summary, we developed a new type of molecular imaging
20 agent based on the cancer targeting peptide cRGDfK coupled to an
aminoacid functionalised NDI. Fluorescence lifetime mapping,
fluorescence intensity profile, lifetime components and lifetime
decay profile were all employed together with MTT assays to
demonstrate the integrity of these molecular imaging agents in
25 cancer cells (PCꢀ3 cells) and their biocompatibility against
cancerous (PCꢀ3) as well as nonꢀcancerous (FEKꢀ4) cell lines. The
close similarity between the lifetimes measured for these
compounds in solution and those determined within cells confirm
the cellular uptake of such molecules, and offer compelling
30 evidence that these are different with respect to those of free,
untreated living cells. These measurements confirm the integrity of
such NDI compounds within living cells and their cytoplasmic
distribution.
We thank the University of Bath and EPSRC for
35 studentships (ZH, JAT, RLA) and the EC (ERC Consolidator
Grant O2SENSE), Royal Society, MRC and STFC for
financial support. We also thank the EPSRC Mass
Spectrometry Service at Swansea for assistance, STFC for
funding the SRS crystallography work, Mr Colin Wright
40 (Nikon Bioimaging Ltd), Prof. Jon Dilworth and Mr Colin
1
2
T. Acker and K. H. Plate, J. Mol. Med., 2002, 80, 562.
(a) C. D. Gadaleta and G. Ranieri, Crit. Rev. Oncol. Hemat.,
2011, 80, 40; (b) A. Lequerrec, D. Duval and G. Tobelem,
Baillieres Clin. Haem., 1993, 6, 711; (c) K. A. Rmali, M. C. A.
Puntis and W. G. Jiang, Colorectal Disease, 2007, 9, 3.
V. Pedchenko, R. Zent and B. G. Hudson, J. Biol. Chem., 2004,
279, 2772.
65
3
70
4
(a) M. A. Buerkle, S. A. Pahernik, A. Sutter, A. Jonczyk, K.
Messmer and M. Dellian, Brit. J. Cancer, 2002, 86, 788; (b) S.
Cressman, Y. Sun, E. J. Maxwell, N. Fang, D. D. Y. Chen and
P. R. Cullis, International Journal of Peptide Research and
Therapeutics, 2009, 15, 49; (c) M.Fani, D. Psimadas, C. Zikos,
S. Xanthopoulos, G. K. Loudos, P. Bouziotis and A. D.
Varvarigou, Anticancer Res., 2006, 26, 431; (d) V. I.
Romanov and M. S. Goligorsky, Prostate, 1999, 39, 108.
75
5 (a) M. A. Dechantsreiter, E. Planker, B. Matha, E. Lohof, G.
Holzemann, A. Jonczyk, S. L. Goodman and H. Kessler, J.
Med. Chem., 1999, 42, 3033; (b) R. Haubner, H. J. Wester, W.
A. Weber, C. Mang, S. I. Ziegler, S. L. Goodman, R.
SenekowitschꢀSchmidtke, H. Kessler and M. Schwaiger,
Cancer Res., 2001, 61, 1781; (c) U. Hersel, C. Dahmen and H.
Kessler, Biomaterials, 2003, 24, 4385.
80
85
Sparrow (University of Oxford) and
Dr John Warren
6 M. Albota, D. Beljonne, J. L. Bredas, J. E. Ehrlich, J. Y. Fu, A.
A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D.
McCordꢀMaughon, J. W. Perry, H. Rockel, M. Rumi, C.
Subramaniam, W. W. Webb, X. L. Wu and C. Xu, Science,
1998, 281, 1653.
(University of Manchester) for training and helpful
discussions.
90
Notes and references
7
(a) S. Asir, A. S. Demir and H. Icil, Dyes and Pigments, 84, 1;
(b) H. E. Katz, J. Johnson, A. J. Lovinger and W. J. Li, J. Am.
Chem. Soc., 2000, 122, 7787.
Z. Hu, G. D. Pantos, N. Kuganathan, R. L. Arrowsmith, R. M.
J. Jacobs, G. KociokꢀKoehn, J. O'Byrne, K. Jurkschat, P.
Burgos, R. M. Tyrrell, S. W. Botchway, J. K. M. Sanders and
S. I. Pascu, Advanced Functional Materials, 2012, 22, 503.
P. A. Waghorn, M. W. Jones, M. B. M. Theobald, R. L.
Arrowsmith, S. I. Pascu, S. W. Botchway, S. Faulkner and J. R.
Dilworth, Chem. Sci., 2013, 4, 1430.
45 # State Key Lab of Heavy Oil Processing, China University of Petroleum-
Beijing, No 18 Fuxue Road, 102249, Beijing,
8
9
¶Department of Chemistry, University of Bath, Claverton Down, BA2
7AY, UK
95
§Department of Pharmacy and Pharmacology, University of Bath,
50 Claverton Down, BA2 7AY, UK
‡Central Laser Facility, Rutherford Appleton Laboratory, Research
Complex at Harwell, STFC, Didcot, OX11 0QX, UK
*Corresponding authors: Dr. S. I. Pascu, E-mail: s.pascu@bath.ac.uk;
Dr G.D.Pantos, g.d.pantos@bath.ac.uk, Dr. Zhiyuan Hu, E-mail:
55 zh209@cup.edu.cn
100
10 A. T. R. Williams, S. A. Winfield and J. N. Miller, Analyst,
1983, 108, 1067.
4
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