Water soluble perylene bisimide and its turn off/on fluorescence are used to detect cysteine and homocysteine

Article abstract of DOI:10.1039/c5nj00608b

Perylene derivatives (P) in the presence of melamine (M) are envisioned as novel fluorescent probe for mercury ions and form a selective complex (PMHg) with mercury ions in neutral medium (pH = 7). This chemo-sensing ensemble probe exhibits high selectivi

Full text of DOI:10.1039/c5nj00608b

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ARTICLETYPE
Water Soluble Perylene Bisimide and its Turn Off/On Fluorescence are
Used to Detect of Cysteine and Homocysteine
Pradip K. Sukul,a Dines C. Santra,a Pradeep K. Singh,b Samir K. Maji b and Sudip Malik a*.
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Fluorescent chemosensors based on “turn on” method have
attracted much attention because of their high sensitivity and high
50 potential in biological imaging. In contrast to covalent
interaction,12 chemoꢀsensing ensemble13 formed through nonꢀ
covalent interactions is considered to be powerful sensing
platform for biothiols. Electrophilic metal cation such as Hg2+ or
Cu2+ are responsible to make chemoꢀsensing ensemble by
55 interacting thiol groups with the metal cations leading to the
dissociation of ensemble.14
Perylene derivatives (P) in presence of melamine (M) have
envisioned as a novel fluorescent probe for mercury ion and
form selective complex (PMHg) with mercury ion at neutral
medium (pH=7). This chemo-sensing ensemble probe exhibits
10 high selectivity and sensitivity for thiol-containing amino
acids, and detects cysteine and homocysteine from healthy to
abnormal levels under physiologically-relevant conditions.
Among twenty amino acids, bio thiols have important role in
maintaining the appropriate reducing environments of proteins,
15 cells, organisms, and act as redox regulator.1 Cysteine (Cys) and
homocysteine (Hcy) are essential low molecular weight amino
thiols that are involved in protein synthesis, detoxification,
metabolism, and required for the growth of cells and tissues in
living systems.2 Increasing levels of Cys are associated with
20 neurotoxicity,3 and Cys deficiency causes many syndromes such
as slow growth rate, hair depigmentation, oedema, skin lesions,
weakness, lethargy, and liver damage.4 Excess of Hcy results a
risk factor for cardiovascular5a and Alzheimer’s disease,5b neutral
tube defects, inflammatory bowel disease, complications during
25 pregnancy, and osteoporosis.5c Therefore, it is necessary to
develop efficient methods for the detection of Cys/Hcy in
physiological media for academic research as well as clinical
applications.6 By taking consideration of strong nucleophilicity of
thiol group, few efforts have been addressed to develop
30 colorimetric and fluorometric detection of Cys/Hcy.7 Several
pathways including the Michael addition,8 cleavage reactions,9
cyclization with aldehydes,10 and others 11 have also been tried to
serve the purpose. In spite of their sensitivities towards thiolꢀ
containing compounds, these probes suffer the key limitations:
35 (a) irreversible due to the formation covalent bond, (b)
interferences from other thiols and (c) not working in the aqueous
medium.
a)
HOOC
O
N
O
O
N
O
COOH
COOH
NH2
60
65
70
N
N
H2N
N
NH2
HOOC
M
P
b)
Hg2+
Cysteine
or
Homocysteine
Fig. 1 (a) Chemical structure of P and M. (b) Schematic illustration for
the sensing of biothiol (Cys, Hcy). In the presence of mercury ions, P of
75 PM12 complex coordinate to Hg2+; upon addition of Cys/Hcy Hg2+ was
extracted and the emission for PM12 complex regained. Concentrations
of PM12: 1 × 10ꢀ5 M; Hg2+: 2 × 10ꢀ5 M; PMHg: 1 × 10ꢀ5 M; Cys: 2 × 10ꢀ5
M; Hcy: 2 × 10ꢀ5 M, in HEPES buffered (pH 7.0) water.
Perylene bisimide derivatives are important chromophores
80 owing to their extensive use as pigments as a result of their high
thermal stability and chemical inertness.15 Because of their high
quantum yield and excellent photostability, these derivatives have
been considered to be the best fluorophores for single molecular
a Polymer Science Unit, Indian Association for the Cultivation of Science,
40 2A & 2B Raja S. C. Mullick Rd., Jadavpur, Kolkata – 700032, India.
b
Department of Biosciences and Bioengineering, IIT Bombay, Powai,
spectroscopy.16 However, most of them are not soluble in water
17
Mumbai-400076, India.
E-mail: psusm2@iacs.res.in.
† Electronic Supplementary Information (ESI) available: Experimental
45 details for the preparation of PM12 solution. Plot of change of emission
intensity, BenesiꢀHildebrand analysis, quantitative estimation of Cys and
Hcy in blood serum and urine sample. See DOI: 10.1039/b000000x
85 due to the presence of large hydrophobic π conjugated ring
.
We
report here waterꢀsoluble chemoꢀsensing probe (PM), composed
of perylene derivative (P) and melamine (M), which can detect
Hg2+ via ‘turn off’ fluorescence.
In the presence of M, P becomes more soluble to form an
90 aqueous mixture PM12 (molar ratio is 1:2, 1 × 10ꢀ5 M) which
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generates very bright yellow luminescence (Fig. 1b). UVꢀVis
containing amino acids (LꢀCys and LꢀHcy) have surprisingly
spectra of PM12 (10 µM) in HEPES buffer exhibit bands at 452 60 recovered the intensity of fluorescence in the solution (Fig. S6).
nm, 499 nm and 536 nm and these are obvious signature of
monomeric P. To check the optical property of PM12, aqueous
solution of individual metal ion (Na+, K+, Mg2+, Co2+, Ni2+, Cu2+,
Ag+, Zn2+, Hg2+, Fe2+, Ca2+, Cd+2 and Pb+2 ions) has been added
Whereas, other amino acids even at 100ꢀfold higher concentration
of Cys and Hcy do not induce any increase in the fluorescence
intensity of the solution. Significantly, other thiol containing
compounds (NꢀacetylꢀLꢀcysteine, glutathione, 3ꢀmercaptoꢀ
5
to the solution of PM12 (Fig. S1).18 Only three metal ions(Cu2+, 65 propanoic acid) do not have any interference (Fig. S7). This
Pb+2 and Hg2+) induce significant changes of the absorption
spectra (Fig. S2), implying their responses to PM12 under the
important result implies the practical application of the detection
of Hcy and Cys quantitatively in the presence of other amino
acids in biological samples, indicating its diagnostic potentiality
of novel PMHg probe.
10 experimental condition. To investigate the binding property of
PM12 toward the Hg2+ ion, titration experiments were carried out
as shown in Fig. S3. On addition of Hg2+ ion to the solution of
PM12, the absorption intensities at 499 nm and 536 nm are
gradually diminished with the generation of new peak at around
15 556 nm and the intensity the peak at 452 nm is more or less
unchanged. These changes are seen up to the addition of 2 equiv
of Hg2+ ion, implying that carboxylic groups in PM12 coordinate
to Hg2+ ion forming a PM12/Hg2+ (PMHg) complex in this case.
The new peak at 556 nm is a characteristic absorption band of the
20 PMHg complex and can be used to diagnose whether the PM12
binds to Hg2+ ion or not.
5x106
70
75
80
85
90
4x105
3x105
2x105
a)
b)
18
0
ꢀM
50 ꢀM
4x106
3x106
2x106
0 ꢀM
ꢀ
M
600
700
800
Wavelength (nm)
600
700
800
Wavelength (nm)
1x105
1x106
0
0
500
600
700
800
500
600
700
800
Wavelength (nm)
Wavelength (nm)
c)
6.0x106
700
a)
New peak
1200
800
400
0
b)
600
500
400
300
200
100
0
0
4.0x106
25
20 ꢀM
600
700
800
2.0x106
0.0
Ag+
Na+
K+ Pb2+ Cu2+ Co2+Ni2+ Fe2+Hg2+
Zn2+ Cd2+Mg2+Ca2+
500
600
700
800
PM12 with metal ions
30
Wavelength (nm)
Fig. 2 (a) Fluorescence titration of PM12 with Hg2+ in HEPES buffer
solution at room temperature ([P] = 1 × 10−5, [M] = 2 × 10ꢀ5 M. (b)
Fluorescence spectra profiles of PM12 (10 µM P) in HEPES buffer
35 solution with various metal ions (20 µM). Inset spectra shows the gradual
generation of new band.
PMHgSer Val Tyr Gly Glu Phe Asp Lys Pro Ala Ile Thr His Trp Cys HcysGTH Arg Met Gln
Amino acids
Fig. 3 Fluorescence titration of PMHg upon addition (a) Cys and (b) Hcy.
Inset spectra show the gradual decrease of charge transfer band [λex = 470
95 nm, path length = 10 mm]. (c) Fluorescence responses of PMHg upon
addition 100 equivalent of various amino acids and peptides.
Fluorescence spectra of PM12 (λexc = 470 nm) show the
characteristic emission band at 551 nm and 590 nm, indicating P
is free from π−π stackings. Gradual addition of Hg2+ to the
40 solution of PM12 (10 µM), the quenching of emission bands is
instantaneously observed (Fig. 2a), it completes at 2 equivalent of
Hg2+ and the association constant of PM12/Hg2+ complex is
6.84x105 Mꢀ1. Except three metal ions (Pb+2, Cu2+ and Hg2+),
flourogenic responses (Fig. S4) of PM12 to other metal ions do
45 not show significant spectral change even in the presence of 100
equiv each of other metal ions. It is noteworthy to mention that
PM12 shows higher fluorescence quenching towards Hg2+ rather
than paramagnetic Cu2+ and Pb+2 (Fig. 2b). Upon addition of Hg+2
ions, ꢀCOOH groups of PM12 become deprotonated, and the
50 resulting perylene moieties get aggregated to show the quenching
of monomeric emission which is compensated by a weak
excimer emission observed at higher wavelength (690 nm, inset
of Fig.2a).
Titration spectra of PMHg with Cys and Hcy in HEPES
buffer at pH 7 (Fig. 3aꢀb) have revealed that with increasing the
concentration of Cys/Hcy, the fluorescence intensity at 551 nm
100 gradually are enhanced (Fig. S8). The remarkable increase of the
fluorescence is also perceived by the naked eye either in normal
light or under UV lamp (Fig. 1b). The UVꢀVis titration
experiment also bears the response of PMHg with Cys or Hcy.
The characteristic emission peaks of PM12 are appearing,
105 concomitantly, excimer emission band (690 nm, inset of Fig.3) is
diminishing. It points out that there is no chemical reaction
between PM12 and Cys/Hcy. The apparent binding constant for
the interaction of PMHg with Cys/Hcy at pH 7 is calculated from
the BenesiꢀHildebrand analysis (Fig. S9, 1.45x104 Mꢀ1 for Cys
110 and 7.46x105 Mꢀ1 for Hcy). Important outcome is that the probe
PMHg is able to detect reversibly both Cys and Hcy at both
healthy and abnormal levels (Fig.S10ꢀS14).
To evaluate the selectivity of nonꢀfluorescence ‘‘chemoꢀ
55 sensing probe’’ (PMHg) towards amino acids or peptides, the
fluorescence studies in HEPES buffer solution have been
performed (molar ratio of PM12:Hg2+ was 1:2 to quench the
fluorescence completely). Among all amino acids, only two thiolꢀ
The switching behaviour of our sensor was further tested
inside the HeLa cells with the use of a confocal fluorescence
115 microscopy. PM12 was not toxic to cells as observed by the MTT
assay (Fig. S15). Brightꢀfield measurement also confirmed the
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viability of cell throughout the experiments. Incubation of HeLa 60 detection of Cys/Hcy over glutathione at physiological pH. This
cells with 10 ꢁM of PM12 for 0.5 h at 37ᵒC produced bright
fluorescence (Fig. 4b). After the cells were subsequently
supplemented with Hg2+ solution (20 ꢁM) for 30 min at 37ᵒC,
complete quenching of fluorescence was noticed (Fig. 4c).
Consequently, HeLa cells were further incubated with Cys for 30
min at 37ᵒC, a remarkable increase of intracellular fluorescence
was observed (Fig. 4d). The images reveal that the fluorescence
signals are localized in the perinuclear/cytoplasmic space of the
10 cells, providing a subcellular distribution of Cys. The study
further indicates that PM12 is membrane permeable, which
makes this sensor valuable for cellular sensing applications.
Moreover, from the bioimaging of cells (Fig. 4c), we have found
that PMHg does not show fluorescence enhancement, suggesting
15 that other biomolecules inside the cellular environment do not
effect the fluorescence property of PMHg suggesting the
specificity of our sensor
approach includes the coordination of Hg2+ with Cys/Hcy. The
fluorescence technique is selectively used to measure Cys or Hcy
in the presence of other biothiols in human blood plasma and
urine.
5
65 Experimental
General:
Reagents were purchased from sigmaꢀaldrich and used as
received and P was synthesized according to our earlier reported
procedure.17 For all aqueous mixtures and for spectroscopic
70 studies, water of HPLC grade was used and for the preparation of
solution for sensing purpose HEPES buffer (pH = 7) were used.
1HꢀNMR spectra were recorded at room temperature on 300 MHz
spectrometers (Bruker). Matrixꢀassisted laser desorption
ionization timeꢀofꢀflight (MALDIꢀTOF) mass spectrometry was
75 carried out with Bruker Daltonics FLEXꢀPC using CHCA as a
matrix. The UVꢀVis spectra of all samples were studied with
HewlettꢀPackard UVꢀVis spectrophotometer (model 8453).
Fluorescence studies for all samples, prepared in a sealed cuvette,
were carried out with Horiba Jobin Yvon Fluoromax 3 instrument
80 at excited wavelength 470 nm and spectra were recorded with
slits 2/2 nm.
c)
a)
b)
d)
20
Fig. 4 Confocal fluorescence images of Hg2+ in Hela cells (Zeiss LSM
Synthesis of N, N’-Di-(phenyl-3, 5-dicarboxylic acid)-perylene-
3,4:9,10-tetracarboxylic diimide(P):17
25 510 META confocal microscope, 40 × objective lens). (a) Brightfield
transmission image of HeLa cells. (b) Fluorescence image of HeLa cells
incubated with PM12 (10 ꢁM). Further incubated with addition (c) 2
equiv HgCl2. (d) Again treated with Cys (2 equiv.). Scale bar on each
image is 50 ꢀm.
2.00g (5.10 mmol) of perylene tetracarboxylic dianhydride,
85 2.308g (12.74 mmol) 5ꢀaminoisophthalic acid and 10g of
imidazole were heated at 130ᵒC for 6 hrs under Ar atmosphere.
Then 150 mL of ethanol was poured into the hot mixture and
refluxed for overnight. Then the reaction mixture was cooled to
room temperature precipitate out. The precipitate was filtered and
90 washed with ethanol (50 mL, 3 times). The product was dried at
70°C under vacuum oven to get deep red powder like pdt. Yield:
30
Encouraged by the sensing behaviour of PMHg in vitro and in
cells, we have measured the detection capability of Cys or Hcy in
human blood plasma as well as urine quantitatively.19 Due to
malꢀfunctioning of the cellular processes, changes in the amount
of low molecular mass (thiols and disulfides) in body fluids are
1
80%. H NMR (300 MHz , D2O) δ ppm: 8.52 (s, 2H ) , 8.14 (s,
35 observed. Analysis of the amount of biothiols is important. As the
concentration of biothiols is very high in both serum and urine,
the biological samples are diluted before the assays performed.
The determined biothiol concentrations are listed in Table S1ꢀ4.
By using the Cys calibration curve as the standard, the unknown
40 endogenous amounts of thiols in diluted urine or diluted serum
have been determined. The calculated thiol values for the
undiluted urine and serum samples by using the Cys calibration
curve show 50 µM and 245 µM respectively. These values
provide the normal range of value in urine and serum. Because of
45 the presence of much higher concentrations of Cys in urine or
serum, we believe this value determined using Cys calibration
curve should be accurate. Recovery of added known amounts of
Cys to the urine or serum samples is from 96 % to 101 %. These
results suggest the potentiality of this probe for thiol
50 determination in practical applications. To further prove the
better quality of our system, we have compared the fluorescence
intensity between the Cys calibration curve and the urine or
serum samples (with original plus spiked Cys), as shown in Fig.
S12aꢀb. The total amount of Hcy content in the commercial blood
55 by similar way as for cysteine is found to be 15 µM (Fig. S14aꢀ
b). It is expected that the sensor can be used in thiol detection for
biological samples.
4H), 7.23 (m, 8H ); IR (KBr) νmax = 746, 808, 851, 1115, 1254,
1355, 1575, 1662, 1696, 2927, 3152 cmꢀ1; MALDIꢀTOFꢀMS m/z
95 calculated for C40H18N2O12: 718.58, found: 741.14 [M+Na+].
UV/Vis (H2O): λmax/nm (ε/Mꢀ1cmꢀ1) 532 (27653), 499 (19416) ,
464 (8476); Fluorescence (H2O): λmax/nm: 551, 594 and
Fluorescence quantum yield, Φf = 0.21.
100 Preparation of stock solution:
PM12 solution was prepared in HEPES buffered solution by
mixing melamine solution (2 x 10ꢀ5 M) to the solution of P (1 x
10ꢀ5 M) in HEPES buffer. The solution was kept at 15ᵒC for 12
hrs to complete the formation of supramolecular polymer. Then
105 the solution was used for the synthesis of the chemosensing
ensemble probe selectively with Hg2+ among all other metal ions.
Stock solutions of Hg2+, GSH, Cys, Hcy and various other amino
acids (1 mM) were prepared by dissolving in water.
For sensing the thiol in urine sample, the urine sample
110 collected from a healthy adult volunteer was used as the urine
stock solution. The test solution was prepared by adding the
requisite amounts of stock solutions together, diluting with pH
7.0 HEPES buffer. The solution was stirred for 30 seconds and
fluorescence spectra were recorded. The final urine concentration
115 in the test solution is 10ꢀfold diluted.20 All experiments related to
blood serum and urine were performed in compliance with the
In conclusion, we have successfully developed
a
supramolecular as well as chemoꢀsensing probe (PMHg) for the
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committee of IACS, Kolkata.
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15
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