Colorimetric and fluorescent dual probe based on a polythiophene derivative for the detection of cysteine and homocysteine

Article abstract of DOI:10.1039/c1cc11990g

Colorimetric and fluorescent dual detection of cysteine (Cys) and homocysteine (Hcy) has been realized based on the ionic self-assembly of a cationic polythiophene derivative with aromatic derivatives of Cys and Hcy formed in situ.

Full text of DOI:10.1039/c1cc11990g

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Colorimetric and fluorescent dual probe based on a polythiophene
derivative for the detection of cysteine and homocysteinew
Zhiyi Yao, Hua Bai, Chun Li* and Gaoquan Shi*
Received 8th April 2011, Accepted 28th April 2011
DOI: 10.1039/c1cc11990g
Colorimetric and fluorescent dual detection of cysteine (Cys) and
homocysteine (Hcy) has been realized based on the ionic self-
assembly of a cationic polythiophene derivative with aromatic
derivatives of Cys and Hcy formed in situ.
2-cyano-6-methoxybenzothiazole (CBT) can readily react with
Cys and Hcy in phosphate buffered saline (PBS) at room
temperature with fast kinetics and a high conversion rate,
leading to the formation of luciferin derivatives.12 The luciferin
derivatives with a large aromatic plane and negative charge
can form a complex with cationic PMTPA through ionic self-
assembly (ISA).13 The assembly process can be easily
monitored by recording the absorption and emission spectral
changes originating from the changes of the PT conformation
and aggregation state. Therefore, colorimetric and fluorescent
dual detection of Cys and Hcy can be established.
Low-molecular-weight thiols, such as cysteine (Cys), homo-
cysteine (Hcy) and glutathione (GSH), play crucial roles in
many biological processes in organisms. Abnormal levels of
Cys or Hcy are related to many diseases, such as slowed
growth, Alzheimer’s disease and cardiovascular disease.1
Thus, it is important to develop efficient methods for the
detection and quantification of biothiols in physiological
media for academic research and clinic applications. Up to
now, lots of strategies have been designed to detect Cys and
Hcy, for instance high performance liquid chromatography
and capillary electrophoresis separations coupled with different
detection methods,2 gas chromatography–mass spectrometry,3
electrochemical assays,4 optical detection based on the inter-
plasmon coupling of Au nanorods,5 nanoparticles,6 quantum
dots7 and synthetic receptors.8–11 Among the various
strategies, optical assays based on synthetic colorimetric and
fluorescent probes have attracted increasing interest during the
last decade because of their high sensitivity, selectivity and
simplicity.11 The fluorescent method is favoured due to its low
detection limit and potential for in vivo imaging of living cells,
whereas the colorimetric protocol possesses inherent merits,
which include visual detection and real-time in situ responses,
which are suitable for the analysis of biological samples
in vitro.11 Although lots of optical probes have been synthesized
and applied to the detection of Cys and Hcy, examples of
colorimetric and fluorescent dual probes for the detection of
Cys and Hcy are still rare.10,11
Cationic PMTPA can form complexes with various
bioanalytes through non-covalent interactions,14 however, its
interaction with structurally simple bioanalytes, such as amino
acids and peptides, is rather weak.15 To enhance its interaction
with Cys, an in situ reaction of Cys with CBT was performed
in PBS (137 mM NaCl, 2 mM KCl, 10 mM phosphate buffer,
pH 7.4) containing 5% (v/v) DMSO.16 Upon mixing CBT and
Cys in PBS, CBT–Cys, a stable aromatic derivative of Cys was
formed with fast kinetics, reaching a conversion rate of 80%
within 30 min (see ESIw, Fig. S1). The formation of the
CBT–Cys conjugate was confirmed by 1H NMR (see ESIw,
Fig. S2) and mass spectral (MS) analysis (MALDI-TOF MS
(m/z): 316.98 (M + Na)+).
Herein, we report a selective and sensitive colorimetric and
fluorescent dual method for the detection of Cys and Hcy
based on the assembly of a polythiophene (PT) derivative,
poly(3-(4-methyl-30-thienyloxy)propyltrimethylammonium)
(PMTPA). The design rationale for the optical dual
detection is depicted in Fig. 1. Commercially available
Department of Chemistry, Tsinghua University, Beijing 100084,
China. E-mail: chunli@mail.tsinghua.edu.cn, gshi@tsinghua.edu.cn;
Fax: +86-10-6277-1149; Tel: +86-10-62798909
w Electronic supplementary information (ESI) available: Experimental
procedures and additional figures. See DOI: 10.1039/c1cc11990g
Fig. 1 (a) The chemical structure of PMTPA and the reaction scheme
for the in situ reaction between CBT and Cys (or Hcy) in PBS. (b) The
ISA process of PMTPA in the presence of CBT and Cys (or Hcy).
c
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Chem. Commun., 2011, 47, 7431–7433 7431

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(see ESIw, Fig. S3). It was found that, upon the introduction
of these bioanalytes, most of the PMTPA solutions remained
yellow with lmax o 462 nm, except for those containing Cys
and Hcy, which gave a purple solution (Fig. 3). Therefore, one
can expect that cationic PMTPA could be used as a colorimetric
probe for the detection of Cys and Hcy by combining the
in situ reaction with CBT. The ratio of the absorbance
difference between those at 590 nm (p-stacked aggregates)
and 700 nm to the absorbance at 450 nm (PMTPA random
coil), (A595 ꢀ A700)/A455, was calculated to further estimate the
selectivity of PMTPA toward Cys and Hcy (Fig. 4). It is
clearly seen that the most striking effect was observed for
Cys and Hcy, whereas the other analytes do not obviously
influence the absorbance of PMTPA. Most notably, the ratio
values in the presence of Cys and Hcy are 24 times higher than
those in the presence of GSH, a major interferent in most Cys
and Hcy sensing systems. These results indicate that PMTPA
is selectively responsive to Cys and Hcy.
To check the viability of ratiometric detection of Cys, the
variation in the absorbance spectra of PMTPA with increasing
amounts of Cys in PBS buffer was examined (Fig. 5). It was
found that upon addition of increasing amounts of Cys, the
absorption maximum is gradually red-shifted from 455 to 595 nm,
with a dramatic colour change from yellow to purple. Notably,
there exists a good linear correlation between (A595 ꢀ A700)/
A455 and the Cys concentration (R = 0.995 from 0.05 to
0.25 mM), indicating that this approach is applicable to the
ratiometric detection of Cys. To further verify the feasibility of
the present approach, the detection of Cys in PBS buffer
containing excess GSH (0.5 mM) was examined.18 In this case,
a certain known amount of Cys (0.1 and 0.125 mM) was added
to PBS buffer and the concentration was determined using a
suitable calibration curve. The percentage recovery was
determined to be 114% and 90%, respectively. These results
indicate that this protocol would be useful in rapid semi-
quantitative or qualitative detection of Cys.
Fig. 2 Absorption (a) and CD (b) spectra of PMTPA (0.1 mM) in the
absence and presence of CBT, Cys and the mixture of CBT and Cys
(CBT–Cys) in 10 mM PBS buffer containing 5% DMSO. [CBT] =
[Cys] = 0.25 mM.
Fig. 2 compares the absorption and CD spectra of PMTPA
and its mixtures with CBT, Cys and the CBT–Cys conjugate
formed through the procedures described above. PMTPA
exhibits an absorption maximum at 455 nm in PBS buffer
(pH 7.4), which is attributed to a random-coiled conformation
of the PT backbone.14 Upon addition of CBT–Cys (the
mixture of CBT and Cys) to the PMTPA solution, the
absorption maximum is red-shifted and features two major
peaks at 548 and 595 nm and a shoulder at around 510 nm,
which are characteristics of the formation of a p-stacked
supramolecular aggregate.17 Simultaneously, a distinct colour
change of the solution from yellow to purple was observed.
The formation of PMTPA superstructures induced by the
addition of CBT–Cys was further confirmed by CD examination
(Fig. 2b). PMTPA does not have a chiral centre, thus, it is
intrinsically optically inactive and no CD pattern in the p–p*
transition region can be detected. Interestingly, the addition of
CBT–Cys into the PMTPA solution induced an intense CD
signal in the p–p* transition region of PMTPA. However,
CBT or Cys alone can scarcely induce the spectral changes
of PMTPA. These observations indicated that the luciferin
derivative with an aromatic moiety formed in situ in PBS
buffer solution greatly improved the ability to induce
aggregation of the PMTPA chains from a random-coiled
conformation to a chirally ordered phase.17
It is known that conformational changes and aggregation
play crucial roles in controlling the emissive properties of
conjugated polyelectrolytes. The yellow, random-coiled form
of PMTPA is fluorescent, exhibiting an emission at 575 nm
upon excitation at 495 nm. It was found that upon increasing
the amount of Cys, the emission intensity of PMTPA at 575 nm
was gradually quenched (Fig. 6). These spectral results indicate
that fluorometric detection of Cys is also possible, and the
detection limit can be extended to the order of 10ꢀ10 M.
To evaluate the specificity of PMTPA toward Cys and Hcy
based on the in situ derivatization and ISA, the absorp-
tion spectra of the PMTPA solution upon addition of
CBT, 20 natural amino acids and some sulfur-containing
bioanalytes, including GSH, cysteine (Cyt) and g-L-glutamyl-
L-cysteine (GC), were examined under identical conditions
Fig. 3 The colorimetric response of PMTPA (0.1 mM) in 10 mM
PBS buffer containing 5% DMSO induced by the addition of
CBT (0.25 mM) and various analytes. [Cys] = [Hcy] = 0.25 mM;
[other analytes] = 0.5 mM.
c
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Fig. 4 The relative absorbance of PMTPA (0.1 mM) in the presence
of CBT (0.25 mM) and 20 natural amino acids, Hcy, Cyt, GSH and
GC. [analytes] = 0.25 mM.
Fig.
6 (a) The variation in the emission spectra of PMTPA
(0.01 mM) in PBS buffer containing 5% DMSO with increasing
concentrations of CBT–Cys as indicated. [CBT] = 2[Cys]. (b) Fluorescence
quenching of PMTPA by CBT–Cys at various concentrations. I0 is the
fluorescence intensity at 575 nm of a solution of PMTPA; I is
the fluorescence intensity at 575 nm of a solution of PMTPA in the
presence of different amounts of CBT–Cys. Excitation wavelength:
495 nm.
Fig. 5 The variation in the absorption spectra of PMTPA (0.1 mM)
in PBS buffer containing 5% DMSO with increasing concentrations of
Cys, as indicated. [CBT] = 0.25 mM.
In conclusion, we have developed a novel approach for
colorimetric and fluorescent dual detection of Cys and Hcy in
aqueous media based on an in situ derived reaction and ionic
self-assembly of PMTPA. Interference from amino acids and
certain biothiols is minimal. This method shows great promise
for the colorimetric and fluorescent detection of thiols in
biofluids.
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This work was supported by the National Natural Science
Foundation of China (50973054, 91027028, 50873052).
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Chem. Commun., 2011, 47, 7431–7433 7433