Dual-functional probe based on rhodamine for sequential Cu2+ and ATP detection in vivo

Article abstract of DOI:10.1016/j.saa.2018.06.094

A rhodamine-based fluorescent probe for Cu²⁺ and ATP has been designed. The fluorescence intensity/absorbance was significantly enhanced upon the addition of Cu²⁺ owning to the opening of the spiro-ring of rhodamine, which quickly returned to the original level due to the reconstruction of the probe by the reacting with ATP. Cu²⁺/ATP-induced fluorescent intensity/aborbance changes showed a good linear relationship with the concentration of Cu²⁺/ATP in the range of 2–20 μM/0–10 μM with a detection limit of 0.1 μM/1.0 μM. The proposed method is simple in design and fast in operation, and is suitable for the reversible monitoring of Cu²⁺ and ATP in bioanalytical applications.

Full text of DOI:10.1016/j.saa.2018.06.094

Accepted Manuscript
Dual-functional probe based on rhodamine for sequential Cu2+
and ATP detection in vivo
Xilang Jin, Jingkai Gao, Pu Xie, Mengchen Yu, Ting Wang,
Hongwei Zhou, Aijie Ma, Qian Wang, Xin Leng, Xianghan Zhang
PII:
S1386-1425(18)30636-X
doi:10.1016/j.saa.2018.06.094
SAA 16253
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 22 February 2018
Revised date:
Accepted
date:
23 June 2018
2
5 June 2018
Please cite this article as: Xilang Jin, Jingkai Gao, Pu Xie, Mengchen Yu, Ting Wang,
Hongwei Zhou, Aijie Ma, Qian Wang, Xin Leng, Xianghan Zhang , Dual-functional
probe based on rhodamine for sequential Cu2+ and ATP detection in vivo. Saa (2018),
doi:10.1016/j.saa.2018.06.094
This is a PDF file of an unedited manuscript that has been accepted for publication. As
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ACCEPTED MANUSCRIPT
2
+
Dual-functional probe based on rhodamine for sequential Cu and
ATP detection in vivo
‡
,a
‡,b
a
c
a
a,∗
a
Xilang Jin , Jingkai Gao , Pu Xie , Mengchen Yu , Ting Wang , Hongwei Zhou , Aijie Ma ,
a
d
b,∗
Qian Wang , Xin Leng , Xianghan Zhang
‡
Xilang Jin and Jingkai Gao contributed equally to this work.
a
School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an
710032, Shaanxi China
b
School of Life Sciences and Technology, Xidian University, Xi’an 710071, Shaanxi China
State and Local Joint Engineering Lab. of Advanced Network and Monitoring Controls，
c
Xi'an Technological University, Xi'an 710032, Shaanxi China
d
Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule
,
Chemistry, College of Chemistry & Materials Science, Northwest University, Xi an, Shaanxi
710069, PR China
∗Corresponding author at: School of Materials and Chemical Engineering, Xi'an
Technological University, Xi'an 710032, Shaanxi China
Tel.: +86 029 86173324; Fax: +86 029 86173324. E-mail: jinxilang_911@163.com
Abstract: A rhodamine-based fluorescent probe for Cu2+ and ATP has been designed. The
fluorescence intensity / absorbance was significantly enhanced upon the addition of Cu2+
owning to the opening of the spiro-ring of rhodamine, which quickly returned to the original
2
+
level due to the reconstruction of the probe by the reacting with ATP. Cu /ATP-induced
fluorescent intensity/aborbance changes showed a good linear relationship with the

ACCEPTED MANUSCRIPT
2+
concentration of Cu /ATP in the range of 2-20 µM/ 0-10 µM with a detection limit of 0.1µM
/
1.0 µM. The proposed method is simple in design and fast in operation, and is suitable for
2+
the reversible monitoring of Cu and ATP in bioanalytical applications.
2
+
Key words: rhodamine, fluorescence, ATP, Cu , bioimaging
1
. Introduction
As the third most plentiful essential trace metal (after iron and zinc), copper
performs a crucial role in carrying out several necessary biological processes in plants
and animals, including humans. However, excessive or inadequate intake of copper
would result in several serious human afflictions, such as Wilson’s disease, Menkes
syndrome and Alzheimer’s diseases [1]. In addition, Adenosine triphosphate (ATP), as
one of the most indispensable biological molecules, plays vital roles in a series of
biological processes including energy metabolism and signaling, DNA replication and
transcription. Deficiency in the ATP level caused many diseases, such as Parkinson’s
diseases and hypoglycaemia [2]. Thus, development of highly sensitive and selective
methods for real-time monitoring of the concentration level of Cu2+ and ATP is very
conducive to explore the physiological mechanism of Cu2+ and ATP in vivo and
related disease diagnosis.
Among the various reported detection methods, the fluorescence bioimaging
technology has become a powerful tool for detecting and monitoring biologically
active species (e.g., anions, metal ions, ROS, RNS, RSS) [3] in vitro and in vivo by
virtue of the superiorities of operational simplicity, high sensitivity, great temporal and

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spatial resolution [4-5]. Among the various fluorophores, rhodamine scaffold has
attracted increasing interest in biomolecular detection due to its excellent
photophysical characteristics, such as high fluorescence quantum yield, long
wavelength absorption and emission and great photostability [6]. Currently, numerous
rhodamine-based probes have been reported to detect various individual species [7],
3
+
2+
2+
3+
2+
2+
3+
e.g. metal ions (Fe [8], Cu [9], Hg [10], Au [11], Zn [12], Pd [13], Al [14]),
-
2-
reactive sulfur species (SCN [15], S [16], Cys [17]), reactive oxygen and nitrogen
[18], O -
[19], ClO [20], ONOO [21], NO [22]) and ATP [23].
-
-
species (H
2
O
2
2
Unfortunately, the fluorescence probes that could be applied to sequential detect Cu2+
and ATP in the literatures are still rare, which encourages us to develop a
2
+
dual-functional rhodamine-based probe for real-time monitoring both Cu and ATP in
vivo.
In this study, we successfully designed and synthesized a novel rhodamine
spirolactam-based probe (Scheme 1). The probe shows turn-on fluorescent and colorimetric
2+
response for Cu and turn-off response for ATP with high sensitivity and selectivity.
Scheme 1
2
. Experimental
2
.1 Materials and apparatus
All metal nitrate salts or hydrochloride salts, rhodamine B, 4-tert-Butylphenol,
hexamethylenetetramine and anhydrous trifluoroacetic acid were commercially
purchased from Energy Chemical Scientific, Co., Ltd (Shanghai China) and used as
received. Double distilled water was used throughout all the experiments. Fluorescent

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spectra were measured on a Hitachi F-4500 fluorescence spectrophotometer equipped
with a xenon discharge lamp. UV-Vis spectra studies were obtained with Shimadzu
UV-170 spectrphotometer. High resolution mass spectra (HRMS) were recorded on a
Brukermicro TOF-Q
Ⅱ ESI-Q-TOF LC/MS/MS Spectrometer by means of the
electronic spray ionization (ESI). All nuclear magnetic resonance (NMR) spectra were
1
recorded on a Varian INOVA-400 MHz spectrometer (at 400 MHz for H NMR and
1
3
1
00 MHz for C NMR) using tetramethylsilane (TMS) as internal standards. Athymic
nude mice (4-6 weeks old weighing 20-25 g) were commercially purchased from
Beijing HFK Bioscience Co. Ltd., China. In vivo fluorescence imaging analysis was
carried out in an IVIS Kinetic imaging system.
2.2 General Procedure
The stock solution (1 mM) of probe
1 was prepared by directly dissolving in
+
+
+
acetonitrile. The stock solutions (1mM) of various metal nitrate salts (Li , Na , K ,
2
+
2+
2+
+
2+
2+
2+
4+
2+
2+
2+
3+
2+
2+
3+
Ba , Ca , Cd , Ag , Mg , Co , Mn , Sn , Pb , Hg , Ni , Cr , Cu , Fe , Fe ,
2
+
-
-
-
-
2-
-
-
-
2-, SO
2-, NO -
, NO -
Zn ) and anions (F , Cl , Br , I , CO
PO
-, PO 3-, HPO 2-, PPi, AMP, ADP) were dissolved in distilled water and diluted
to the desired concentrations. During the titration experiments, different quantities of
3
, HS , HSO
3
, Ac , S
2
O
3
4
2
3
,
H
2
4
4
4
2
+
Cu solutions and 0.10mL probe
1 (1000 µM) were mixed and filled up with PBS to
1
0 mL in volumetric tubes. During the interference experiments, 0.020 mL of 1000
2
+
µM Cu , 0.100 mL of 1000 µM probe
1 and 0.100 mL of 1000 µM testing species
were mixed and filled up with PBS to 10 mL in volumetric tubes. Complex 1-Cu2+
solution for detection of anions were prepared via adding different amounts of Cu2+

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ions (20 µM) to probe
1 (10 µM) solution and then were used to recognize related
anions (100 µM). The titration experiments and interference experiments of ATP is
2
+
similar to that of Cu . All fluorescence spectra were recorded 5 min after the addition
of determinant at 25 °C. 1 mL aliquots were pipetted into a 1 cm cuvette for spectral
measurements. The slit width was 5 nm for both excitation and emission.
2
.3 Synthesis of intermediate 3
Hexamethylenetetramine (4.77g, 34.0 mmol) and 4-tert-Butylphenol (2.55g, 17.0
mmol) were dissolved in anhydrous trifluoroacetic acid (30 mL) under N
reaction mixture was refluxed at 80 °C for 24 h. The mixture was poured into 4 M HCl
100 mL) and stirred for 10 min, after which it was extracted with CH Cl (2×100 mL).
The combined organic layers were washed with 4 M HCl (2×100 mL) and water (100
mL), then dried over anhydrous Na SO and concentrated under vacuum. The crude
2
. The
(
2
2
2
4
product was purified by column chromatography, eluting with CH
viscous liquid (1.85 g, 61.06%).
2
Cl to afford yellow
2
2
.4 Synthesis of probes 1
Rhodamine B hydrazide was prepared according to the reported procedure in
literature. [24]
Rhodamine B hydrazide (1.00 mmol, 0.456 g) was mixed with compound 3 (1.10
mmol, 0.196 g) in 30 mL ethanol and then heated at refluxed for 8 hours. After cooling
to room temperature, the solvents were removed under reduced pressure and the
residue further purified by column chromatography using MeOH: CH
2
Cl
2
= 1: 100 as
1
eluent to give 0.420 g pure probe
1
as pink power in yield of 68.19%. H NMR(CDCl ,
3

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4
00 MHz): δ (ppm) 1.18 (t, J=8.8 Hz,12H),1.26 (d, J=9.8 Hz, 9H), 3.35 (q, J=6.8 Hz,
8
H), 6.29 (m, 2H), 6.51 (m, 4H), 6.83 (d, J=8.8 Hz, 1H), 7.16 (d, J=2.4 Hz, 1H), 7.25
(
m, 2H), 7.57 (m, 2H), 8.01 (d, J=7.2 Hz, 1H),9.54 (s, 1H), 10.54 (s, 1H). MS (ESI)
+
m/z = 617.4208 [M+ H] , calc. for C28
H
20
N
2
6
O H = 617.3486.
3
. Results and discussion
3
.1 The recognition properties of probe 1 towards Cu2+
The colorimetric and fluorescent responses of probe
1 with various potential
interfering cations were carried out in CH
3
CN-PBS (1/99, v/v, pH 7.4) solution. The
free probe showed colorless and very weak fluorescence (Φ=0.0012). As shown in
1
Figure 1, the experiments were carried out by comparing the change of the
fluorescence intensity or absorbance before and after the addition of 100 µM other
+
+
+
2+
2+
2+
+
2+
2+
2+
4+
representative species (Li , Na , K , Ba , Ca , Cd , Ag , Mg , Co , Mn , Sn ,
2
+
2+
2+
3+
2+
3+
2+
Pb , Hg , Ni , Cr , Fe , Fe , Zn ). The solution remained no noticeable changes
in emission and absorption profiles in the presence of an excess of other tested analytes
(
100 µM). However, after treatment with Cu2+ (20 µM) under identical condition, a
conspicuous enhancement of fluorescence intensity (at 575 nm) and absorbance (at 552
nm) with a fast response (2 min, Figure S1) were observed. The changes might be
attributed to the copper-induced ring-opening of probe
xanthenes moiety of rhodamine. Furthermore, the competitive experiments showed
that the colorimetric and emission responses of probe
towards Cu2+ were not
considerably interfered in the presence of other related competing metal ions (10.0
equiv.). Hence, the results clearly demonstrated that probe had excellent selectivity
1 and the generation of
1
1

ACCEPTED MANUSCRIPT
2
+
2+
for Cu in the mixtures of other related species, which was highly desirable for Cu
detection in living systems.
Fig. 1
Furthermore, the titration experiments were conducted to examine the sensitivity
2
+
of probe
1 for Cu . The emission / absorption spectra of probe 1 with gradual
concentrations of Cu2+ (0.0-40.0 µM) were recorded (Figure 2). The fluorescence
intensity at 575 nm (absorbance at 552 nm) was enhanced gradually with increasing
2
+
the concentrations of Cu , and up to a 127-fold/141-fold enhancement in fluorescence
intensity/ absorbance (Φ=0.35). Moreover, the fluorescence intensity at 575 nm was
linearly proportional to the concentrations of Cu2+ within the range of 2.0-20.0 µM
(
Figure 3). The regression equation was y = 57.2x + 115.8 (R² = 0.998) and the
detection limit (based on 3δ/k) was calculated to be 0.1 µM. Similarly, a good linearity
2
+
existed in a plot of absorbance at 552 nm and the concentrations of Cu in the 2.0-20.0
µM range with an excellent linear correlation (R² = 0.997). The regression equation
was y = 0.085x - 0.023. These results indicated that probe
1 had a high sensitivity and
sufficient ability for the qualitative and quantitative detection of Cu2+ under
physiological conditions.
Fig. 2
Fig. 3
In order to study practical applicability of the probe, the appropriate pH range for
the probe to function was further investigated. As illustrate in Figure S2, the free probe
itself showed weak fluorescence and stable at pH 4.8-13.0. Moreover, the responses of

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probe
1
toward Cu2+ showed distinct changes over the same range. Fortunately, the
fluorescence response showed exceptional constant in the pH range of 6.8-8.0, which
should be highly favourable for detecting Cu2+ without interference from pH effects
and works well under in the physiological pH range.
2
+
3
.2 The recognition properties of complex 1-Cu towards ATP
Subsequently, we investigated the effects of ATP on the reaction equilibrium
between complex 1-Cu2+ and ATP. As illustrated in Figure 4, the absorption and
fluorescence emission gradually decreased upon the addition of ATP (0.0-1.0 equiv),
and the solution could be changed almost the same as the original probe
1. The
reversible reactions between probe
1
and Cu2+ or ATP could be repeated with good
reproducibility and stability at least four times. A linear calibration graph of the
responses of luminescence intensity/ absorbance at 575 nm/552 nm to the
concentration of ATP (0.0-10.0 µM) was observed (Figure 5). The regression equation
was y = -61.957x + 705.95 (fluorescence intensity) / y = -0.073x + 0.8367
2
(
absorbance) with a linear coefficient R =0.9947/0.9855. The detection limit (LOD)
was calculated to be 1 µM for ATP. The distinct fluorescence signal change indicated
that complex 1-Cu2+ could be potentially employed to detect ATP quantitatively.
Fig. 4
Fig. 5
To validate the selectivity of complex 1-Cu2+ in practice, various potential
-
-
-
-
2-
-
-
-
2-, SO
2-, NO -
,
interfering species, such as F , Cl , Br , I , CO
3
, HS , HSO
3
, Ac , S
2
O
3
4
2
NO , PO
3
-, H PO - 3-, HPO 2-, PPi, AMP, ADP were tested. As shown in Figure 6, these
2
4
4
4

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interfering species did not induce significantly fluorescence/absorption signal changes
to the complex 1-Cu2+ under the same addition. Only upon the addition of ATP,
noticeable fluorescence/absorption quench change was observed. The ATP-induced
quenching of the fluorescence/absorption of complex 1-Cu2+ was also observed,
suggesting a reversible spirolactam ring open-close mechanism. In addition, the
interference experiments were also carried out by comparing the fluorescence/
absorption changes of the solution in the presence of other test species at relative high
concentration (100 µM). The fluorescence/absorption was not influenced by the
coexistence of other substance tested. Therefore, the results illustrated that complex
1
-Cu2+ had specific response towards ATP over other coexisted analytes in
physiological conditions.
Fig. 6
In addition, the complexation / decomplexation cycles could be repeated at least 5
times with good reproducibility and stability with less fluorescent efficiency/
absorbance (Figure 7). The results revealed that the binding of probe 1 and Cu2+ or
ATP is a reversible spirolactam ring open-close mechanism.
Fig. 7
2
+
3
.3 Sensing mechanism study of probe 1 with Cu and ATP
The Job’s plot analysis (Figure S4) exhibited a maximum fluorescence/
2
+
absorption value when the molar fraction of the [1] versus [1] + [Cu ] was 0.5,
2
+
indicating the complexation ratios between probe 1 and Cu was 1:1.

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The proposed sensing mechanism of the probe
1
is shown in Scheme 2. In
accordance with the 1:1 stoichiometry, the probe was the most likely to chelate with
metal ions via its oxygen atoms in carbonyl and phenolic hydroxyl groups. To
2
+
investigate the mechanism responsible for the detection of Cu /ATP by probe
/complex 1-Cu2+, the assay solution of the complex 1-Cu2+ and complex 1-Cu2+
treated with ATP was subjected to high resolution mass spectroscopy (HRMS)
1
2
+
analysis. Upon the addition of Cu , a new peak was obtained at m/z 678.3089
(
1-Cu2+), coinciding exactly with that for the transformation from probe
1 to complex
-Cu2+ (calcd: m/z 678.2620) induced by Cu (Figure S7). When complex 1-Cu2+ was
2+
1
+
treated with ATP, a dominate peak at 617.4217 (M+H ) that is observed to arise which
is in good accordance with that calculated (m/z 617.3489) for probe (Figure S8).
1
That is to say that this complex exhibits response to ATP based on the copper complex
ensemble displacement mechanism.
Scheme 2
Furthermore, IR spectra of probe
respectively in KBr disks, and the results were shown in Figure 8. In the IR spectra, the
peak at 1693 cm-1 of the probe
, which corresponds to the characteristic amid
1
and complex 1-Cu2+ were also checked
1
carbonyl absorption, was shifted to 1649 cm for the complex 1-Cu2+, suggesting that
-1
a strong binding of the carbonyl group occurs with copper ion. The phenolic hydroxyl
-
1
2+
absorption that appeared at 3302 cm disappeared by addition of Cu , confirming that
a strong binding of the phenolic hydroxyl group occurs with copper ion.

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Fig. 8
2
+
3
.4 Detection of Cu in real water samples
To evaluate the applicability of the new probe in practical sample, we employed
probe
1
to determine the concentration of Cu2+ in tap water and pool water samples
from Xi'an Technological University. The water samples were spiked with the
different concentration of standard Cu2+ solutions (1.00 µM, 5.00 µM, 10.0 µM)
respectively, with the results shown in the Table 1. It was observed that the results
obtained for the water samples shown satisfactory recovery values, which clearly
demonstrated that the proposed probe was capable of the detection of Cu2+ in real
samples.
Table 1
2
+
3
.5 Visualization of Cu in vivo
For living cell imaging, biocompatibility is always the first property to examine.
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in
Hela cells (Figure S9) was performed to assess the cytotoxicity of the probe . The
results showed that cell viability is not obviously affected even when the concentration
of probe as high as 10 µM in the culture medium.
Based on the desirable results in fluorescence detection, the potential application
of probe in living animal bioimaging was explored. As shown in Figure 9, the control
group which was given a skin-pop injection of probe showed almost no fluorescence.
The mice injected with Cu2+ (1.0 equiv.) displayed a strong fluorescence and the
fluorescence intensity increased with time, indicating that probe could respond to
1
1
1
1
1

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2
+
Cu . Subsequently, when the mice were treated with ATP, the fluorescence intensity
decreased with time. With the above results taken together, it could be concluded that
2
+
probe
1
could be used to image Cu and ATP in living organisms.
Fig 9
4
. Conclusions
In conclusion, we had successfully presented an “off-on-off” rhodamine-based
2
+
probe for Cu and ATP, which was based on the opening and closing of spiro-ring of
2
+
rhodamine. The probe
1 was highly sensitive and specific for the Cu and ATP in the
reversible cycle detection. Furthermore, it was applied for real water sample analysis
and fluorescence imaging in vivo. We believe that this novel probe could be applied
for not only environmental monitoring but also biological detection in the near future.
Acknowledgements
The work was supported by the National Natural Science Foundation of China (No.
5
1603164, 51608412), the Natural Science Foundation Research Project of Shaanxi
Province (No. 2016JQ2034, 2018JQ2015), the president Foundation of Xi'an
Technological University (No. XAGDXJJ16010).
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Figure caption
Scheme 1 The synthesis of the probes
Fig. 1 Selectivity of fluorescence intensity (λex = 510 nm) (a) and absorption (b) of probe
Cu2+ (20 µM) in PBS buffer (20 mM, pH 7.4, 1 % CH
CN) solution. The pillars in the front
1
.
1
for
3
row represent the value in the presence of various ions. The pillars in the back row indicate the
change in the emission intensity upon subsequent addition of Cu2+ (20 µM) to the solution
containing probe
1 and the respective ion of interest. The fluorescence intensity was measured
at 575 nm. The absorbance was measured at 552 nm.
Fig. 2 Fluorescence intensity (λex = 510 nm) (a) and absorption (b) changes of probe
upon gradual addition of Cu2+ (0.0-40.0 μM).
1 (10 μM)
Fig. 3 The linear response curve of fluorescence intensity (λex = 510 nm) (a) and absorption (b)
in dependence of Cu2+ concentrations. The fluorescence intensity was measured at 575 nm. The
absorbance was measured at 552 nm.
Fig. 4 Fluorescence intensity (λex = 510 nm) (a) and absorption (b) changes of complex 1-Cu2+
(
10 μM) upon gradual addition of ATP (0.0-1.0 equiv).
Fig. 5 The linear response curve of fluorescence intensity (λex = 510 nm) (a) and absorption (b)
in dependence of ATP concentrations. The fluorescence intensity was measured at 575 nm. The
absorbance was measured at 552 nm.
Fig. 6 Selectivity of fluorescence intensity (λex = 510 nm) (a) and absorption (b) of complex
1
-Cu2+ for ATP (10 µM) in PBS buffer (20 mM, pH 7.4, 1 % CH
COCH ) solution. The pillars
3 3
in the front row represent the value in the presence of various ions. The pillars in the back row
indicate the change in the emission intensity upon subsequent addition of ATP (10 µM) to the

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solution containing complex 1-Cu2+ and the respective ion of interest. The fluorescence
intensity was measured at 575 nm. The absorbance was measured at 552 nm.
3
Fig 7. Stepwise complexation / decomplexation cycles carried in CH CN-PBS (1/99, v/v, pH = 7.4)
solution. The fluorescence intensity was measured at 575 nm. The absorbance was measured at 552
nm.
Scheme 2 Proposed mechanism of reversible structure change toward Cu2+ and ATP.
Fig 8. IR of probe 1 and complex 1-Cu2+
Fig 9. Bioluminescence imaging in athymic nude mice.

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Table 1 Detection results of Cu2+ in water samples by probe 1
Cu2+ recovered meana ± SDb
Sample
Cu2+ spiked (μmol/L)
Recovery (%)
(
μmol/L)
0.00
1.00
5.00
10.0
0.00
1.00
5.00
10.00
Not detected
1.03± 0.01
4.84 ± 0.03
9.72 ± 0.08
Not detected
0.98± 0.02
5.11 ± 0.03
10.12 ± 0.07
-
103.0
96.8
97.2
-
pool water
98.0
102.2
101.2
tap water
a
Mean of three determination
SD: standard deviation.
b

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Highlights
An “off-on-off” rhodamine-based probe for Cu and ATP, which was based on the opening
2
+
►
and closing of spiro-ring of rhodamine.
2
+
►
Cu /ATP-induced fluorescent intensity/aborbance changes showed a good linear
2
+
relationship with the concentration of Cu /ATP in the range of 2-20 µM/ 0-10 µM.
►
The proposed method is simple in design and fast in operation, and is suitable for the
2+
reversible monitoring of Cu and ATP in bioanalytical applications.

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9