Highly promising discrimination of various catecholamines using ratiometric fluorescence probes with intermolecular self-association of two sensing elements

Article abstract of DOI:10.1039/c5ra10321e

Two sensing elements based on fluorescence probes have been employed as the promising discriminating sensors of two catecholamines, dopamine (DA) and norepinephrine (NE), acting as a proper guest linker between two self-recognition sensing components. Surprisingly, in the presence of epinephrine (EPI), sensor NB containing boronic acid connected to a napthalimide unit demonstrated a very strong fluorescence enhancement while a large fluorescence quenching was observed in the case of DA and NE. To differentiate the structural similarity of DA and NE, an appropriately designed small fluorescence sensor CC containing a crown-ether attached to coumarin showed complementary recognition to an ammonium ion based catecholamine. The combination of NB and CC is capable of the differentiation of DA and NE with dual emission bands under a PET mechanism. The dual emission ratio (I₄₇₅/I₃₈₄) of the NB-DA-CC complex showed different values from those of the NB-NE-CC complex. Additionally, the PCA analysis using mixed sensors of NB and CC obviously separated DA and NE better than a single sensing element. This systematic approach is the first report showing a high potential for the identification of DA and NE using ratiometric fluorescence sensors with dual emission by two sensing elements.

Full text of DOI:10.1039/c5ra10321e

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Highly Promising Discrimination of Various Catecholamine by
Ratiometric Fluorescence Probes under Intermolecular Self-
Association of Two Sensing Elements
Received 00th January 20xx,
Accepted 00th January 20xx
YanisaSanguansub,a
DOI: 10.1039/x0xx00000x
VithayaReaugpornvisuti,aThawatchaiTuntulani,aVinichPromarak,bBoosayaratTomapatanageta*
www.rsc.org/
Two sensing elements based on fluorescence probes have been employed as a promising discriminating sensor (NB) of
two catecholamines, dopamine (DA) and norepinephrine (NE), acting as a proper guest linker between two self-
recognition sensing components. Surprisingly, in the presence of epinephrine (EPI), sensor NB containing boronic acid
connecting to a napthalamide unit demonstrated a very strong fluorescence enhancement while its large fluorescence
quenching was observed in the case of DA and NE. To differentiate the structural similarity of DA and NE, an appropriately
designed small fluorescence sensor of CC containing crown-ether attached to coumarin showed a complementary
recognition to an ammonium ion based catecholamine. The combination of NB and CC is capable of classification of DA
and NE with dual emission bands under PET mechanism. The dual emission ratio (I475/I384) of NB-DA-CC complex showed
the different values from that of NB-NE-CC complex. Additionally, the PCA analysis using mixed sensors of NB and CC
obviously separated DA and NE better than a single sensing element. This systematic approach is a first report permitting a
high potential for identification of DA and NE by ratiometric fluorescence sensors of dual emission by two sensing
elements.
fluorescent quenching (CHEQ) effect upon binding with
catecholamines.7 Concerning the discrimination of each
catecholamine, many reports regarding fluorescent chemosensors
Introduction
Catecholamine neurotransmitters, including dopamine (DA),
norepinephrine (NE) and epinephrine (EPI), which are a class of
biogenic amines and have structural similarity consisting of catechol
and amino groups and play an important role as neurotransmitters
involving in a variety of central nervous system functions.1-2
Generally, an unusual catecholamine concentration in body fluids is
an essential indicator of patients suffering from Parkinson’s disease
showed the non-specific binding among EPI, DA and NE.14 None of
the fluorescence chemosensors for catecholamine sensing
employed the Fluorescence Resonance Energy Transfer (FRET)
process as a tool in detection. Interestingly, the factors that give
influences on the fluorescent intensity change are environmental
condition, probe concentration and instrumental efficiency.15-16
Ratiometric probe can eliminate most or all vagueness by self-
calibration of emission intensities at two wavelengths.17-21 Chen et
al. designed a probe CouMC by connecting a coumarin fluorophore
and an indolenium block through an ethylene group to address a
hybrid fluorophore of coumarin and merocyanine.22 Addition of HS-
to interact at merocyanine moiety in the probe CouMC altered the
conjugated system and an internal charge transfer (ICT) resulting in
a ratiometric sensing behavior by decreasing merocyanine emission
band and increasing coumarin emission band. Canary’s group23
reported the ratiometric displacement approach to detect Cu2+ by
two fluorophore elements which demonstrated the different
emission upon the different excitation wavelength. A great deal of
works has focused on systematic studies designed to elucidate the
optical properties of the sensory molecules for sensing purpose.
Our previous work24 based on FRET-on process for discrimination of
catecholamine demonstrated the powerful tool for discrimination
of DA and NE from EPI by a FRET-on process induced by an
3-4
in the clinical diagnosis. Such a critically elevated amount of EPI
in the human blood certainly involved the heart failure and the
increase in blood pressure.5 Importantly, a large amount of EPI and
NE in blood and urine possibly causes the patients to suffer from
tumors of adrenal glands.6 Specific detection of each catecholamine
species is a challenging task for sensing purpose. Detection of
specific
catecholamine,
neurotransmitters,
by
artificial
chemosensors under fluorescence spectroscopy7-11 which is a
versatile technique with high sensitivity, rapid response, and easy
performance, is currently of interest subject to pursue. Regarding
the optical detection, there are
a few reports regarding
chemosensor of catecholamine,12 particularly, containing boronic
acid and aldehyde for covalently binding with catechol and amine
groups of catecholamine species to form a boronate ester and an
iminium ion, respectively.13 However, most fluorescent
chemosensors previously reported the chelation-enhanced
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catecholamine neurotransmitter in biological system. Furthermore,
the PCA analysis is utilized for classification of biogenic amine
analytes by fluorescence spectral data. Additionally, we also apply
this conceptual sensing in human urine samples.
intermolecular assembly of coumarin aldehyde (CA) and
pyreneboronic acid (PBA) with catecholamines as a guest linker.
However, this conceptual design showed a well-specific detection
of DA and NE. Taking such a definition of well-selective
discrimination of the similar structure as the particular challenges
for the chemist, the discrimination of similar structure biogenic
amines prompts us to develop a more effective sensory system to
solve this weak stand point. Deep considering on the structures of
DA and NE, NE consists of the additional hydroxyl group addressing
Experimental
General
Nuclear magnetic resonance (NMR) spectra were recorded on
Varian Mercury 400 MHz and Bruker 500 MHz nuclear resonance
spectrometers (in CDCl3, DMSO-d6 and D2O). ESI HRMS spectra were
recorded on Varian Cary Eclipse spectrofluorometer.
on
β-position of side chain. As anticipated, this hydroxyl group
close to ammonium ion of NE might give a different binding affinity
with crown-ether moiety compared to DA without a hydroxyl group
on the side chain. Taking on board the idea of intermolecular self-
assembly in a spontaneously controllable manner, we have
engineered a host-guest complex by suitable two sensing elements
which enable to covalently bind with catechol and interact with
ammonium unit based catecholamine. Apart from interesting
properties of fluorescent sensors, a dual fluorophore giving two
different emission responses by the same excitation wavelength
gave fascinating sensing properties.25-30 Our motivation is to design
two fluorescence chemosensors: i) NB contains boronic acid
connecting to a napthalamide unit for a covalent binding with
catechol and as a fluorophore giving the emission band at 381 nm,
respectively, ii) CC consists of crown-ether unit connecting to
coumarine for non-covalent interaction with ammonium ion based
catecholamine and as a fluorophore with emission band at 475 nm,
respectively.
All materials and solvents were purchased from Aldrich, Fluka,
Merck and TCI as standard analytical grade and used without
further purification. Commercial grade solvents such as acetone,
dichloromethane, methanol and ethanol were purified by
distillation before using. Thin-layer chromatography (TLC) was
performed on silica and alumina gel plates (Kieselgel 60 F254, 1mm,
Merck). Dimethyl sulfoxide as AR grade used in fluorescence
measurement was used without further purification. The synthesis
of N-(1,8-Naphthaloyl)-3-aminophenylboronic acid (NB) was carried
out according to the reported procedure. 31
.
Synthesis of sensor CC
The 2-hydroxymethyl-18-crown-6 (0.151g, 0.515 mmol) and
triethylamine (0.36 mL, 2.575 mmol) in benzene (15 mL) were
stirred at room temperature for 30 min under nitrogen
atmosphere. Then, the reaction mixture was added into a solution
of coumarine acid chloride (2) in benzene (5 mL) by using cannula
and refluxed for 24 hours under nitrogen atmosphere. The solvent
was removed under reduced pressure. The crude product was
purified by column chromatography (Al2O3) with 10% EtOH:CH2Cl2as
1
eluent to obtain sensor CC as a brown oil (0.171 g, 62%). H-NMR
CDCl3 δ 8.35 (s, 1H, ArH), 7.29 (d, J = 8.8 Hz, 1H, ArH), 6.54 (dd, J =
3.47 Hz, 1H, ArH), 6.36 (d, J = 1.6 Hz, 1H, CCH), 4.39-4.24 (m, 2H,
OCH2), 3.61-3.35 (m, 22H, OCH2), 3.38 (q, J = 7.07 Hz, 4H, CH2CH3),
1.19 (t, J = 7.1 Hz, 6H, CH2CH3). Elemental Analysis: Calcd. for
C27H39NO10: C, 60.32; H, 7.31; N, 2.61, found: C, 60.21; H, 7.47; N,
2.42.
Complexation studies by fluorescence spectrophotometry
Typically, a stock solution of 1 x 10-4 M of sensor NB in DMSO was
prepared. All of biogenic amines (DA, NE, EPI, TY, Glu, Lys, His and
Hist) were prepared at concentration of 2 x 10-3 M in 0.01 M.
Scheme 1 Conceptual hypothesis under dual fluorescence response
mechanism
We hypothesized that the suitable catecholamine guest will allow phosphate buffer, pH 7.4. Fluorescence spectra were recorded from
the well-defined linkage between NB and CC to provide different
recognition pattern resulting in the discrimination of DA and NE as
the conceptual illustration shown in Scheme 1. It is well-known
that most fluorophores were quenched by catecholamine group
due to electron rich catechol group. The different quenching
patterns of two fluorophores upon binding with an analyte would
give a different ratiometric fluorescence according to different
350-800 nm at ambient temperature. For fluorescence titration, the
solution of guests was added directly to 2.00 mL of 1 x 10-5 M of
sensor NB in a 1-cm quartz cuvette by a micropipette and the
portion of the mixture solution was stirred for 5 min prior to the
measurement.
binding affinity.
This approach can possibly discriminate
Principle component analysis (PCA) method
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shift from 8.68 to 8.35 ppm and other aromatic protons presented
the upfield shift possibly caused by the electron donating of
methylene bridge as shown in Fig S8 in the ESI. In addition, the 13C-
NMR spectrum showed ester group signals at 163.97 and 158.54
ppm. HR-ESI mass spectrum showed the intense peak of m/z at
560.251 corresponding to the structure of CC. (Fig. S10 in the ESI).
For PCA analysis, the solution of guests (100 equiv.) were added to
the solution of sensor NB (1 x 10-5 M) and stirred for 5 min. Then,
0.1 mL of 15-crown-5 was added to the solution mixture and stirred
for 10 min. The solution of sensor CC (5 x 10-5 M) was added in the
same 1-cm quartz cuvette by a micropipette and stirred for 5 min.
The fluorescence spectra were monitored after each addition. The
complexation was repeatedly detected for 5 times for each guest.
The evaluation of PCA was calculated from the responsive
fluorescence spectra in the range of 350 to 800 nm using MATLAB
7.11 (version R2011a) program.
Complexation study of sensor NB with EPI in human urine sample
A stock solution of 1 x 10-4 M of sensor NB was prepared in DMSO.
The stock solution of 2 x 10-3 M of EPI analyte was prepared in 0.01
M phosphate buffer pH 7.4 and the synthetic urine was prepared in
milli-Q water as listed in Table S1 in the ESI. For calibration curve,
the synthetic urine was spiked into the 1 x 10-5 M of sensor NB
solution by the 100-fold diluted solution of urine. The EPI solution Scheme 2 Synthesis pathway of sensor CC
was added to the mixture solution as various concentrations
(10-7μM) and stirred for 5 min. The fluorescence spectra were
Complexation studies of sensor NB with various guests by
recorded from 350-800 nm at the excitation of 340 nm. Therefore, a
calibration curve was built from the plot of I-I0 at the emission
wavelength of 490 nm versus concentration of EPI.
fluorescence spectrophotometry
The sensor NB contains boronic acid moiety as a binding unit for
condensation reaction with catechol group of analyte and
napthalimide moiety as a fluorophore. The selectivity of sensor NB
was examined in biological system (1:9 v/v of DMSO:phosphate
buffer at 0.01 M, pH 7.4) toward 100 equiv. of various biogenic
amines including dopamine (DA), norepinephrine (NE), epinephrine
(EPI), tyramine (TY), L-glutamic acid (Glu), L-lysine (Lys), histidine
(His) and histamine (Hist).The chemosensor NB showed the
characteristic emission band at 384 nm. As similar to previous
reports, most of chemosensors upon complexing with
catecholamine underwent the PET process since electron transfer
from phenyl based catecholamine to napthalimide moiety causes
the fluorescence quenching. Likewise, sensor NB in the presence of
DA and NE showed the fluorescence quenching. On the contrary,
sensor NB covalently bound with EPI surprisingly exhibits a strong
fluorescence with a concomitant of a large red shift from 384 nm to
490 nm as shown in Fig. 1. This finding suggests that after
complexation, the combination of donating ability of the secondary
amine based EPI and a poor acceptability of boronic ester in NB-EPI
adduct extremely enhances the totally orientation of dipole
moment to acceptor part of napthalimide resulting in the
predominant ICT state (Intramolecular Charge Transfer) as shown in
Fig. 1. In the case of DA and NE, the PET process is more
dominative than the ICT process because the complex of sensor NB
with DA and NE gives a slight influence on their dipole moments. On
the other hand, other analytes without the catechol group including
TY, Glu, Lys, His and Hist could not induce the fluorescence changes
implying that boronic ester did not form on sensor NB.
Then, the 100-fold diluted solution of urine samples was spiked
into the 1 x 10-5 M of sensor NB solution. The 40 μM of EPI solution
was added to the mixture solution and stirred for 5 min. The
manipulation was repeated for three times. The fluorescence
spectra were recorded from 350-800 nm at the excitation of 340
nm. After that, the found amount and percent recovery were
calculated from the calibration curve.
Results and discussion
Molecular sensors were designed and synthesized for sensitive and
selective detection of catecholamines through dual fluorescence
responses under one excitation wavelength. The structure of
catecholamines consists of two functional groups, catechol and
amino group. Therefore, the designed sensor should contain the
binding sites for the selective binding with each part of
catecholamine guests.
Synthesis of Sensor CC
The sensor NB was prepared in a single-step reaction by a
nucleophilic substitution reaction between 3-aminophenylboronic
acid hemisulfate and 1,8-naphthalene dicarboxylic acid anhydride in
56% yield. Sensor CC was obtained by a coupling reaction of 2 and
2-hydroxymethyl-18-crown-6 by nucleophilic substitution using
triethylamine as base (as shown in Scheme 2). From the NMR
spectrum of CC, a singlet proton of coumarin showed the upfield
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Figure 2 The fluorescence titration spectra of sensor NB (1 x 10-5 M)
upon gradual addition of (A) DA, (B) NE and (C) EPI at 0-80 equiv. in
1:9 v/v DMSO: phosphate buffer at 0.01 M, pH 7.4 (λex = 340 nm)
The fluorescence intensity at 384 nm of the complexation of sensor
NB toward DA, NE and EPI gradually decreased upon the increment
of catecholamine guests as illustrated by Fig. 2(A), (B) and (C),
respectively. Interestingly, the new emission band at 490 nm of
complexation NB-EPI was significantly developed. Fluorescence
titration data were analyzed by using non-linear regression plot
(Fig. S13) to provide the log Ks values of complex NB with DA, NE
and EPI of 4.13, 4.17 and 4.01, respectively. All guests employed the
catechol group to covalently bind with boronic acid based NB. It can
be reasonably explained that the ammonium ion on DA and NE
preferred to promote the condensation reaction of boronic acid and
catechol group producing a negative boronate ester. It is reliably
rationalized that the log Ks values of sensor NB with DA and NE is
slightly higher than the sensor NB with EPI.
Figure 1. Fluorescence spectra of sensor NB (1 x 10-5 M) in 1:9 v/v of
DMSO:phosphate buffer at 0.01 M, pH 7.4) in the presence of
various guests at 100 equivalent and inset shows the fluorescence
spectrum of sensor NB and EPI.
It is particularly important to extensively investigate the visual
detection of EPI observing from a large fluorescence change and
strong fluorescence intensity. To verify the selective detection of
NB towards various analytes by naked-eyes sensing, the solution of
NB in the presence of various analytes was exposed to 356 nm UV-
visible light. The fluorescence color changes of sensor NB toward
analytes were displayed in Fig. 3. The solution of sensor NB in the
Regarding to the fluorescence changes of sensor NB in the presence
of catecholamines (DA, NE and EPI), it is indicative of the selective
binding between sensor NB and catecholamines. To evaluate the
binding mode between sensor NB and catecholmines, the
stoichiometry of complexes was measured by the Job’s method
using fluorescence technique showing the stoichiometry of 1:1
binding mode for sensor NB with DA, NE and EPI (as shown in Fig.
S12 in the ESI)
presence of DA or NE showed
luminescence. On the other hand, the solution of NB-EPI adduct
exhibited a highly green luminescence. Among other guests
a very low brightness of
including TY, Glu, Lys, His and Hist, sensor NB solution still remained
unchanged.
Binding Properties of sensor NB with DA, NE and EPI by
fluorescence spectrophotometric titration technique
To verify the binding affinity of sensor NB, fluorescence titration of
sensor NB was carried out in phosphate buffer solution pH 7.4.
Figure 3 Fluorescence responses of (A) sensor NB (1 x 10-5 M) in 1:9
v/v DMSO:phosphate buffer at 0.01 M, pH 7.4) in the presence of
the various guests 100 equiv. and Fluorescence images (B) of (a)
sensor NB and (b) The filter paper dipped in sensor NB solution, and
the word “EPI” with EPI solution was exposed to the UV light.
Furthermore, to develop the EPI sensor on the solid support, the
filter paper was dipped in 5 x 10-5 M of sensor NB solution. Then,
this filter paper was painted the word “EPI” using a paintbrush
dipped in EPI solution. The prepared filter paper exposed to 356 nm
UV light, showed a strong green luminescence in word “EPI” as
illustrated in Fig. 3B. As a result of fluorescence changes on solid
support, sensor NB offers an excellently promising detection of EPI
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on the paper base. The results also implied that the sensor NB can
bind with EPI yielding the NB-EPI adducts on the solid support.
(a)
Determination of detection limit of sensor NB with DA, NE and EPI
by fluorescence spectrophotometry
The detection limits of sensor NB toward DA, NE and EPI measured
in a range of 11.9-95.2 ꢀM, 13.0-130.0 ꢀM and 5.0-95.0 ꢀM were
7.71, 8.50 and 1.54 µM, respectively. As consistent with a large
fluorescence change of NB-EPI complex, sensor NB can serve as an
effective EPI sensing in a very low concentration compared to DA
and NE. (Fig S15 in the ESI)
Complexation studies of sensors NB and CC with various guests by
fluorescence spectrophotometry technique
(b)
Although sensor NB offers considerable promises as EPI selective
fluorescence probes, our further purpose is to discriminate the
similar structure of DA and NE. Therefore, we developed
ratiometric fluorescence probes using two fluorescence sensory
elements (NB and CC) containing the different binding sites and
employed intermolecular self-assembled complex to give different
emission bands upon the same excitation wavelength. We
hypothesized that the adaptive self-assembled complexation of
boronic acid based NB and crown-ether based CC by an induced fit
of a proper guest linker, DA or NE, would perform the different
recognition patterns as a consequence of the different fluorescence
responses
of
dual
fluorophores.
Notably,
naphthalimidefluorophore containing boronic acid and crown-ether
coumarin exhibit the different emission bands at 384 and 475 nm,
respectively, upon excitation at 340 nm. Since the structures of DA
and NE are different in the side chain, we expect that the different
ratio of dual fluorescent responses after complexation could be
obtained.
Figure 4. Fluorescence spectra of (a) mixing sensors NB (1 x 10-5 M)
and CC (5 x 10-5 M) (b) sensor CC (5 x 10-5 M), in the presence of
various guests 100 equiv. in 1:9, v/v DMSO: phosphate buffer (0.01
M, pH 7.4) with 0.1 mL 15-crown-5 (λex = 340 nm)
Sodium cation in phosphate buffer solution could interfere the
recognition affinity of ammonium ion of guest with crown-ether
based CC. To overcome this problem, the competitive receptor for
Na+ ions was utilized. From the previous report, the binding
constant of 15-crown-5 with Na+ ion is much higher than that of 15-
crown-5 with ammonium ion.32 For all manipulation, the
complexation study of CC system in buffer solution will add the 15-
crown-5 to preferentially bind with Na+ ions.
Upon the addition of DA or NE, the fluorescence intensity of both
sensors was quenched possibly caused by PET process from phenyl
donor group to napthalimide and coumarin acceptor group as
shown in Fig. 4. Compared to the emission band at 475 nm of
sensor CC with various analytes, the emission band at 384 nm of
complex NB with DA along with NE showed a larger quenching
while other guests did not effect to the spectral change. This
suggested that napthalimide obtained a stronger effect of energy
transfer from guests than coumarin in the case of DA and NE. The
emission band at 475 nm of CC for NE exhibited a larger decrease
(I0-I = 100 a.u.) than that for DA (shown in green and red lines,
respectively, in Fig. 4(a)). It should be noted that the spectra of
sensor CC with guests showed a large quenching only with NE with
(I0-I = 40 a.u.) and no change of emission band for CC and DA
(shown in green and red lines, respectively, in Fig. 4(b)). This
implies that NE could bind more strongly with sensors NB and CC
than DA because the hydroxyl group of NE may undergo the
complementary hydrogen bonding interactions with crown-ether
To verify the selectivity of dual sensory system, the
complexation of sensors NB and CC toward 100 equiv. of various
guests including DA, NE, EPI, TY, Glu, Lys, His and Hist was evaluated
in 1:9 v/v DMSO:phosphate buffer at 0.01 M, pH 7.4). In Fig. 4(a),
the observation of the fluorescence changes of mixed sensors NB
and CC upon adding DA, NE, and EPI signified a well-specific
recognition of dual sensory system towards catecholamines. Other
biogenic amines with the two sensory system showed a small
change of both emission bands indicating non-fitted complexation
for both sensing elements.
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based CC. For better understanding of the binding behaviors, responses upon complexation, PCA method was reliably utilized to
reduce the dimensionality of data set for easier interpretation.
Due to the highly selective binding affinity of sensor NB with EPI as
previous mentioned result, we have examined the clustering
discrimination of other biogenic amines under excluding EPI.
structures of the adduct of NB⊂NE⊂CC have been calculated by
using density function theory (DFT) at the B3LYP/6-31+G(d) level.
Figure 5 exhibited the complementary hydrogen bonding (HB1 and
HB2) between OH based NE and oxygen based napthlimide with
hydrogen based crown-coumarine, respectively, and electrostatic
force of -NH3+ and negative charge of boronate ester. This evidence
clearly supports a strong binding of mixed sensors NB and CC with
NE resulting in a larger fluorescence quenching at 475 nm
compared to single sensing element of CC. Definitely, the mixing of
sensors NB and CC enables to enhance the binding affinity of CC
with DA and NE.
Figure 6. PCA score plots of (A) sensor NB and (B) sensors NB and
CC upon addition of various guests (100 equiv.) in 1:9 v/v DMSO:
phosphate buffer at 0.01 M, pH 7.4) and excluding fluorescence
data of EPI for determining of PCA analysis
Figure 5.DFT/B3LYP/6-31−G(d) optimized structures of the induced
fits of NB-guest-CC complex.
Two dimensional score plot for two principal components (PC1 and
PC2) represents 94.70% variance of the classification of biogenic
amines as shown in Fig 6. The appearance of catecholamine clusters
including DA and NE on the border line between quadrant I and
quadrant III was separated from other biogenic amines while, the
cluster of Hist and TY appeared in quadrant II. This implied that the
sensor NB can bind slightly with Hist and TY. Cross-validation LDA of
this model showed 77.5% accuracy of discrimination for 7 biogenic
amines.
Owing to a well-classified determination, this approach offers very
promising features for a discriminate-sensing purpose for DA and
NE. These indicated that DA and NE act as a proper guest linker
between NB and CC.
As advantage of different fluorescence responses upon
complexation, the self-coordination between sensors NB and CC
with the bridging analytes of DA or NE induced the different
ratiometric fluorescence intensity of coumarin and napthalimide
(Icoumarin/Inapthalimide) as shown in Fig. 4a (inset). As a result, the ratio
of dual emission of complex NB-DA-CC of 4.84 showed a slight
larger than that observed for complex NB-NE-CC (R = 4.28).
Fantastically, the ratiometric fluorescence changes of each complex
still allowed the constant ratio upon the different concentration of
sensors and guest as well as the different condition in the presence
of excess amount of guest.
In our further purposes to separate the similar structures of DA
and NE, the fluorescence spectra of mixing sensors NB and CC
toward biogenic amines were classified by using PCA analysis. Two
dimensional score plot for PC1 and PC2 representing 96.81%
variance was illustrated in Fig. 6b. This PCA model demonstrated
that two sensing elements of sensors NB and CC could clearly
differentiate the similar molecules of DA and NE in quadrant I and
quadrant III, respectively. Hence, both sensors give a high
performance for discrimination of DA and NE while, Hist cluster
appeared in quadrant II closed to the cluster of sensors and other
biogenic amines. Moreover, cross-validation LDA exhibited 77.5%
accuracy of discrimination for all 7 biogenic amines. The PCA score
Principle component analysis (PCA) method for analysis of
complexation33
Furthermore, to easily identify various biogenic amines by using the
different recognition patterns in terms of the different fluorescent
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plot of two sensing elements of NB and CC definitely gave a higher Figure 8. PCA score plots of mixed NB and CC upon addition of
discrimination of DA and NE than a single sensing element of NB.
various guests (100 equiv.) in urine samples and excluding
fluorescence data of EPI for determining of PCA analysis
Complexation study of sensor NB with EPI in human urine sample
Many techniques have been devoted for determination of DA
To develop fluorescence sensor NB in the analytical application, we shown in Table S2. These methods provide ultrahigh sensitive
applied the detection of EPI in real biological system of human urine approach. It can be seen from Table S2 that most of the detection
samples. A linear range of 10-70 µM was obtained by plotting the limits obtained by fluorescence, electrochemistry and colorimetric
intensity of fluorescence at 490 nm versus the spiked various method are lower than micromolar range. Although the detection
concentration of EPI in the synthetic urine as indicated in Fig. S19 in limit of our sensory system is not better than that found in the
the ESI. The spiked urine samples were prepared by the 100-fold previous reports, the normal amount of DA in human serum should
diluted solution of urine by standard addition method. The % be in the range of 10-7-10-5 M.34 For outstanding results of our
recovery for EPI at 40 µM of the spiked solution is in the range of sensory system, an effective discrimination of DA and NE are
100.14 to 101.06% which are the acceptable recovery in sensing definitely remarkable and fantastic which is a rare report focusing in
application as displayed in Table 1. Therefore, the fluorescence this important approach due to quite similar structure of guest.
sensor NB can serve as an effective sensing of EPI in the analytical This aspect is still challenge for chemists.
application of real samples.
Conclusions
Table 1 Results for the determination of EPI in human urine
samples
An effective discrimination of EPI, DA and NE has been
achieved by the development of ratiometric fluorescence of
samples
spiked
(µM)
40
found
(µM)
recovery a
(%)
two sensing elements of NB and CC
discriminate from other biogenic amines with
.
EPI was clearly
strong
a
emission band at 490 nm. Ratiometric fluorescence sensors
NB to CC displayed the emission ratio (I490/I384) of NB-DA-CC
and NB-NE-CC complexes as 4.84 and 4.28, respectively, at the
excess amount of guest. We have also successfully reported
the obviously different recognition pattern of two sensors NB
and CC toward catecholamines by PCA method. For analytical
application, chemosensor NB offers a good result for detection
of EPI in human urine samples. This is the first example of the
significant discrimination of DA and NE by intermolecular self-
assembled recognition of two sensors. This potential research
highlights the promising concept of intermolecular ratiometric
fluorescence sensors using two sensing elements.
human urine-1
human urine-2
a mean ±std, n = 3
40.05± 1.09
40.42± 1.74
100.14± 2.74
101.06± 4.34
40
Furthermore, the discrimination of catecholamine in urine samples
was carried out by PCA method as show in Fig 7. Two dimensional
score plot for two principal components (PC1 and PC2) represents
96.74% variance of the classification of biogenic amines. The
clusters of DA and NE appeared in quadrant I and quadrant II,
respectively, which was remarkably separated from other biogenic
amines. This PCA plot of urine samples is consistency with that in
Fig 6. It satisfied that this dual emission system of mixed sensors NB
and CC highlights an effective discrimination of DA and NE in clinical
diagnosis.
Acknowledgements
We gratefully acknowledge the Thailand Research Fund (TRF),
Commission on Higher Education (CHE) (RSA5680015 and
RTA5300083), the Ratchadaphiseksomphot Endowment Fund
of Chulalongkorn University (RES560530126-AM) and the 90th
Anniversary of Chulalongkorn Fund (Ratchadaphiseksomphot
Endowment Fund) for research grants.
Notes and references
‡
Road, Bangkok, 10330
aDepartment of Chemistry, Faculty of Science, Chulalongkorn University, Phyathai
*Corresponding author: Fax 662-2541309
E-mail: tboosayarat@gmail.com
Electronic Supplementary Information (ESI) available: this article containing
synthesis, NMR spectra, Mass spectra, Fluorescence spectra and Job’s plot analysis.
DOI: 10.1039/x0xx00000x
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