A facile approach for sensitive, reversible and ratiometric detection of biothiols based on thymine-mediated excimer-monomer transformation

Article abstract of DOI:10.1039/c2cc32064a

A ratiometric fluorescent sensor was developed for detecting biothiols in water and biological samples such as urine and serum. For this sensor, the monomer-excimer emission variations were regulated by aggregation and deaggregation via the complexation between pyrene-thymine and mercury ions modulated by biothiols.

Full text of DOI:10.1039/c2cc32064a

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A facile approach for sensitive, reversible and ratiometric detection of
biothiols based on thymine-mediated excimer–monomer transformationw
Boling Ma,a Fang Zeng,*a Xizhen Lia and Shuizhu Wu*ab
Received 19th January 2012, Accepted 25th April 2012
DOI: 10.1039/c2cc32064a
A ratiometric fluorescent sensor was developed for detecting
biothiols in water and biological samples such as urine and serum.
For this sensor, the monomer–excimer emission variations were
regulated by aggregation and deaggregation via the complexation
between pyrene–thymine and mercury ions modulated by biothiols.
The sensing element was obtained simply by linking a fluorescent
dye (pyrene) to thymine, which is one of the four nucleobases in the
nucleic acid of DNA. On the one hand, pyrene monomer molecules
(emission at 350–400 nm) are known to form intramolecular
excimers (emission at 450–500 nm) due to aggregation, and the
excimer could return to the monomer state via deaggregation,12,13
and this particular property has been employed to design
fluorescent sensors.12d,13d On the other hand, thymine (T)
has been demonstrated to be one of the most specific ligands
for Hg2+ that can form a T–Hg(II)–T complex with a strong
affinity and high selectivity.14 Hg(II) has a very high affinity for
biothiols, and the formation constant for the Hg(II)–thiol
complex has been estimated to be over 30 orders of magnitude
greater than that for the binding of Hg(II) to thymine.15 Thus,
by synthesizing a pyrene–thymine compound (PyT) and then
forming the PyT–Hg(II)–TPy complex, we could bring two pyrene
moieties into close proximity to constitute the pyrene excimers. In
the presence of biothiols, due to the even stronger binding between
mercury ions and biothiols, the PyT–Hg(II)–TPy excimers
were transformed into monomers as a result of extraction of
the mercury ions from the former. This excimer–monomer
transformation induced fluorescence variation and afforded
the complex (PyT–Hg(II)–TPy), a ratiometric sensor for
biothiols, as shown in Fig. 1. The detection system is quite
sensitive (with the detection limit of 69 nM for GSH), and can
be used in biological fluids such as urine and serum.
Biothiols play crucial roles in maintaining biological systems,
among which glutathione (GSH), cysteine (Cys) and homo-
cysteine (Hcy) are the most important low-molecular-mass
aminothiols involved in numerous important functions in
metabolism and homeostasis with similar structures.1,2 GSH
is the most abundant intracellular nonprotein thiol which has
a pivotal role in maintaining the reducing environment in cells
and acts as a redox regulator.2 Cys and Hcy are essential
biological molecules required for the growth of cells and tissues in
living systems.2 Among the biothiol detection approaches, fluores-
cence detection is advantageous in terms of high sensitivity and
simplicity of operation.3 Fluorescent probes capable of detecting
small-molecular-weight biological thiols have received intense
attention recently, and most detection strategies are based on
either the thiols’ strong nucleophilicity or their high binding
affinity towards metal ions.4,5 Most of the thiol fluorescent probes
are based on fluorescence measurement at a single wavelength,
which may be influenced by variations in the sample environ-
ment,6,7 while the ratiometric measurement, which involves
the simultaneous measurement of two fluorescence signals at
different wavelengths followed by calculation of their intensity
ratio, can eliminate the adverse effects on fluorescence signals
and give greater precision to the data analysis relative to single-
channel detection.8,9 So far, a few ratiometric fluorescence
sensors for biothiols have been developed by Lin, Kim and some
other groups.10,11
The pyrene–thymine (PyT) compound was synthesized by a
simple two-step approach, as shown in Scheme S1 (ESIw), and
Herein, we demonstrate a novel and facile strategy for con-
structing a reversible and sensitive ratiometric sensor for biothiols.
a College of Materials Science & Engineering, South China University
of Technology, Guangzhou 510640, P. R. China.
E-mail: mcfzeng@scut.edu.cn; Tel: +86 20 22236363
Fig. 1 Schematic illustration for ratiometric sensing of biothiol (GSH)
based on reversible excimer–monomer transformation. In the presence of
mercury ions, the monomer PyT formed complex (PyT–Hg(II)–Tpy) with
Hg(II); upon addition of GSH, Hg(II) was extracted, and the monomer was
b State Key Laboratory of Luminescent Materials & Devices, South
China University of Technology, Guangzhou 510640, P. R. China.
E-mail: shzhwu@scut.edu.cn; Tel: +86 20 22236262
w Electronic supplementary information (ESI) available: Experimental
section, synthesis schemes, 1H NMR spectra, mass spectra, emission
spectra, absorption spectra, reversibility and antiinterference results,
and suitable pH range. See DOI: 10.1039/c2cc32064a
restored (bluish purple emission). Concentrations: PyT: 1 Â 10À5 M, Hg2+
:
5 Â 10À6 M; PyT–Hg(II)–TPy: 5 Â 10À6 M, and GSH: 1 Â 10À5 M, in
HEPES buffered (pH 7.0) water–DMSO (7 : 1, v:v).
c
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the PyT–Hg(II)–TPy complex was then readily prepared upon
addition of mercury ions. Compound PyT and the complex
PyT–Hg(II)–TPy were well characterized by ESI-MS and
1H NMR (Fig. S1–S4, ESIw).
Fig. S5 (ESIw) shows the fluorescence spectra of PyT
(1 Â 10À5 M) in the absence and presence of mercury ions. In
the absence of Hg2+, the PyT solution exhibited a characteristic
monomer emission of pyrene with peaks at 378 and 397 nm,
and no excimer emission could be observed. Upon addition of
mercury ions, the monomer emission gradually decreased, a
new and broad emission band centered at 473 nm appeared.
This new emission band is attributed to pyrene excimer, and
this is believed to be induced by the formation of the
PyT–Hg(II)–TPy structure, which confined pyrene moieties
within close proximity, as shown in Fig. 1. 1H NMR and
ESI-MS results support this assumption (Fig. S3 and S4, ESIw).
The formation constant for the binding between GSH and
Hg2+ is ca. 1042,15 whereas that for T–Hg–T is only around
106,14b indicating the binding affinity of Hg2+ for GSH is
much greater than that for thymine. Thus, GSH can destroy
Fig. 3 Selectivity of PyT–Hg(II)–TPy (5 Â 10À6 M) toward biological
thiols over various amino acids in HEPES buffered (pH 7.0)
water–DMSO (7: 1, v : v). Column 1: the control; column 2–20: amino
acids (100 mM); column 21–23: Hcy, Cys and GSH, respectively (3 mM).
that the addition of GSH can extract the mercury ions from
the PyT–Hg(II)–Tpy complex and break up the complex into
PyT monomers; while the subsequent addition of mercury ions
can link the PyT into the complex again, without losing
characteristic response, as illustrated in Fig. 1.
the PyT–Hg(II)–TPy structure and form complexes with Hg2+
,
To evaluate the selectivity of this sensor, we investigated the
fluorescence response of the sensor towards different biothiols,
various amino acids and commonly-encountered anions.
From Fig. 3, it is clear that only GSH, Cys and Hcy (3 mM)
showed significant fluorescence variation, whereas other amino
acids (100 mM) even at over 30-fold higher concentration of GSH,
Cys and Hcy did not induce obvious fluorescence change. We also
found that most common anions have no interfering effects either
(Fig. S10, ESIw). Only iodide ion at the concentration of 3 mM has
a slight interfering effect. However, for a healthy human being,
urinary iodide is in the range of 100–200 mg L–1 (0.79–1.58 mM)18a
and blood iodide is about 0.24–1.85 g 10 mL–1 (0.18–1.4 mM);18b
hence in biological samples like urine and serum, iodide should
not cause obvious interference. The effects of some coexisting
substances (like various amino acids and some biologically
abundant cations) on GSH sensing were also determined, as
shown in Fig. S11 (ESIw). One can find that none of the amino
acids or cations causes interferences. We also investigated the
fluorescence response of the sensor towards the three biological
thiols over a concentration range of 0 to 5 mM, and obtained
calibration curves of fluorescence intensity ratio of the sensor vs.
concentration for the three biothiols (Fig. S12, ESIw).
The sensor was also applied to detecting thiols in biological
samples (human urine and fetal bovine serum). Due to high
concentration of thiols in both urine and serum, the samples
are usually diluted before the assays are performed,5d,e and
herein, the determined biothiol concentration values are listed
in Table S1–S4 (ESIw). The unknown endogenous (originally-
existing) amounts of thiols in diluted urine and serum samples
were determined by using the Cys or GSH calibration curves
(Fig. S12, ESIw) as standards, respectively, and the thiol values
for the undiluted urine and serum samples were then calculated
as 25 mM and 200 mM, respectively, by using the Cys calibration
curve; or 25 mM and 160 mM by using the GSH calibration
curve (Fig. S12, ESIw). These values are within the normal
range of biothiol values in urine or serum.19 In consideration of
the much higher concentration of Cys in serum or urine,7b,19a
we think the values determined using the Cys calibration curve
should be relatively more accurate. Recovery of added known
leading to the release of PyT (confirmed by mass spectrum,
Fig. S2B, ESIw). This process causes increase in the distance
between pyrene moieties, and thus decreases the excimer
amount as well as its emission.
Fig. 2 shows the fluorescence spectra of PyT–Hg(II)–TPy
(5 Â 10À6 M) in the presence of different concentrations of
GSH. It can be seen that with the addition of GSH, the excimer
emission gradually decreased, meanwhile the monomer emission
gradually enhanced. Remarkable fluorescence change can be seen
by the naked eye under a UV lamp (Fig. 1). It can be found that
the fluorescence ratio increases with the increasing GSH concen-
tration, and varies from 0.37 to 7.8 when the concentration of
GSH is changed from 0 to 5 mM, corresponding to a high signal-
to-background ratio of 21 : 1. Moreover, the fluorescence intensity
ratio (I378 :I473) as a function of GSH concentration is shown
Fig. 2B and Fig. S7 (ESIw) (the lower concentration part). From
these results, it is obvious that PyT–Hg(II)–TPy could function as a
ratiometric fluorescent sensor for GSH quantification. According
to the literature method,16 the detection limit of GSH was
determined to be 69 nM (Fig. S7, ESIw).
One important feature of an ideal chemosensor is its
reversibility.17 We found that this system exhibited reversible
excimer–monomer transformation upon alternate addition of
mercury ions and GSH, as shown in Fig. S9 (ESIw). We believe
Fig. 2 (A) Fluorescence spectra of PyT–Hg(II)–TPy (5 Â 10À6 M) in
the presence of different amounts of GSH in HEPES buffered (pH 7.0)
water–DMSO (7 : 1, v : v). (B) Fluorescence intensity ratio of
PyT–Hg(II)–TPy (5 Â 10À6 M) as a function of GSH concentration in
HEPES buffered (pH 7.0) water–DMSO (7: 1, v : v). (lexc = 345 nm).
c
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amounts of Cys or GSH to the serum and urine samples was in
general from 91% to 109%, which suggested the potential of the
present method for thiol determination in practical applications.
Moreover, we compared fluorescence intensity ratios between the
Sys (or GSH) calibration curve and the serum or urine samples
(with original plus spiked Cys or GSH), as shown in Fig. S13 and
S14 (ESIw; and one can see that for each specific concentration,
the two values are close to each other, further indicating that the
sensor can be used in thiol detection for biological samples.
The cytotoxicity of PyT–Hg(II)–TPy was evaluated using the
L929 cell line by an MTT assay. Hg2+ ion shows a Grade-IV
toxicity, the highest grade according to United States
Pharmacopoeia and ISO 10993–5 (Fig. S15, ESIw); while the
PyT–Hg(II)–TPy shows a Grade-I toxicity, indicating that the
complex’s toxicity is much lower. Theses results demonstrate
that the sensor herein may possess potential for detecting
thiols in biological fluids in vitro.
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investigated the effect of pH on the fluorescence response of
this system. As shown in Fig. S16 (ESIw), the fluorescence
intensity ratio I378 : I473 of PyT has no significant change from
pH 2.6 to pH 7.4. At pH below 5.0, upon addition of 5 mM
Hg2+, the intensity ratio I378 : I473 remained steady, and the
PyT–Hg(II)–TPy complex could not form. At pH above
5.8 and in the presence of mercury ions, PyT readily formed
the PyT–Hg(II)–Tpy complex. Upon addition of GSH to the
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in the pH range of 5.8–8.4. Therefore, this system could serve
as a ratiometric sensor of thiols in the pH range of 5.8–8.4.
In summary, a fluorescent ratiometric sensing system for
biothiols was successfully developed through a facile approach
based on the monomer–excimer transformation of pyrene
moieties. For this approach, the difference in the binding
affinity between thymine–Hg(II) and biothiol–Hg(II) was employed
to realize the detection. This excimer–monomer emission change
affords the PyT–Hg(II)–PyT, a reversible and sensitive fluorescent
ratiometric sensor for biothiols. Due to the existence of a hydro-
philic thymine moiety, this sensor can be used for biothiol
detection not only in HEPES buffered aqueous solution, but also
in urine and serum; and this ratiometric sensor exhibits a low
detection limit for GSH (69 nM). We suppose that the use of
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We gratefully acknowledge financial support from NSFC
(Project No. 21025415, 21174040 and 50973032).
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c
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Chem. Commun., 2012, 48, 6007–6009 6009