Simple biosensor with high selectivity and sensitivity: Thiol-specific biomolecular probing and intracellular imaging by AIE fluorogen on a TLC plate through a thiol-ene click mechanism

Article abstract of DOI:10.1002/chem.200902505

A handy, specific, sensitive bioprobe has been developed. Tetraphenylethene (TPE) was functionalized by a maleimide (MI) group, giving a TPE-MI adduct that was nonemissive in both solution and the solid state. It was readily transformed into a fluorogen showing an aggregation-induced emission (AIE) property by the click addition of thiol to its MI pendant. The click reaction and the AIE effect enabled TPE-MI to function as a thiol-specific bioprobe in the solid state. Thus, the spot of TPE-MI on a TLC plate became emissive when it had been exposed to L-cysteine, an amino acid containing a thiol group, but remained nonemissive when exposed to other amino acids that lack free thiol units. The thiol-activated emission was rapid and strong, readily detected by the naked eye at an analyte concentration as low as approximately 1 ppb, thanks to the lighting up nature of the bioprobing process. Similarly, the emission of TPE-MI was turned on only by the proteins containing free thiol units, such as glutathione. Clear fluorescence images were taken when living cells were stained by using TPE-MI as a visualization agent, affording a facile fluorescent maker for mapping the distribution of thiol species in cellular systems.

Full text of DOI:10.1002/chem.200902505

FULL PAPER
DOI: 10.1002/chem.200902505
Simple Biosensor with High Selectivity and Sensitivity: Thiol-Specific
Biomolecular Probing and Intracellular Imaging by AIE Fluorogen on a TLC
Plate through a Thiol–Ene Click Mechanism**
Yang Liu,[a] Yong Yu,[a, b] Jacky W. Y. Lam,[a] Yuning Hong,[a] Mahtab Faisal,[a]
Wang Zhang Yuan,[a] and Ben Zhong Tang*[a, b, c]
Abstract: A handy, specific, sensitive
bioprobe has been developed. Tetra-
phenylethene (TPE) was functionalized
by a maleimide (MI) group, giving a
TPE-MI adduct that was nonemissive
in both solution and the solid state. It
was readily transformed into a fluoro-
gen showing an aggregation-induced
emission (AIE) property by the click
addition of thiol to its MI pendant. The
click reaction and the AIE effect ena-
bled TPE-MI to function as a thiol-spe-
cific bioprobe in the solid state. Thus,
the spot of TPE-MI on a TLC plate
became emissive when it had been ex-
posed to l-cysteine, an amino acid con-
taining a thiol group, but remained
nonemissive when exposed to other
amino acids that lack free thiol units.
The thiol-activated emission was rapid
and strong, readily detected by the
naked eye at an analyte concentration
as low as approximately 1 ppb, thanks
to the “lighting up” nature of the bio-
probing process. Similarly, the emission
of TPE-MI was turned on only by the
proteins containing free thiol units,
such as glutathione. Clear fluorescence
images were taken when living cells
were stained by using TPE-MI as a vis-
ualization agent, affording a facile fluo-
rescent maker for mapping the distri-
bution of thiol species in cellular sys-
tems.
Keywords: aggregation-induced
emissions · biological activity · bio-
sensors · tetraphenylethene · thiols
Introduction
assay process offers a number of advantages over other ana-
lytical techniques, such as rapid response, high sensitivity,
low background noise, and a wide dynamic working range.[2]
Thanks to the enthusiastic efforts of scientists devoted to
this area of research, a large variety of fluorescent bioprobes
have been developed.[3] Many of the bioprobes work in a
“turned off” mode; for example, the emission of a fluoro-
phore is switched off when it interacts with a quenching spe-
cies in a biological system through a mechanism of fluores-
cence resonance energy transfer.
The development of fluorescent probes for biomolecular de-
tection has emerged as an active area of research due to its
importance to bioscience and applications in biotechnology
as well as its impact on public health.[1] The fluorescent
[a] Dr. Y. Liu, Y. Yu, Dr. J. W. Y. Lam, Y. Hong, M. Faisal,
Dr. W. Z. Yuan, Prof. Dr. B. Z. Tang
Department of Chemistry
Institute of Molecular Functional Materials
The Hong Kong University of Science & Technology (HKUST)
Clear Water Bay, Kowloon, Hong Kong (China)
Fax : (+852)2358-1594
Cellular thiols are essential biomolecules that play an im-
portant role in biological systems as key components of pro-
tein structures and metabolic intermediates. The thiol mole-
cules are decisive in maintaining appropriate oxidation–re-
duction states of proteins, cells and organisms.[4] Glutathione
(GSH), for example, is a thiol with the highest abundance in
cellular systems. It is a small protein with a short sequence
of Gly-Cys-Glu or GCE that resides in every cell of our
body and is thus vital to life. GSH is so critical to cells that
its deficiency results in cellular dysfunction.[5] Many health
problems, such as Alzheimerꢀs disease, leucocyte loss, liver
damage, psoriasis, cancer, and AIDS are known to be associ-
ated with levels of cellular thiols.[6]
E-mail: tangbenz@ust.hk
[b] Y. Yu, Prof. Dr. B. Z. Tang
Bioengineering Graduate Program, HKUST, Clear Water Bay
Kowloon, Hong Kong (China)
[c] Prof. Dr. B. Z. Tang
Department of Polymer Science and Engineering
Zhejiang University, Hangzhou 310027 (China)
[**] AIE=aggregation-induced emission; TLC=thin-layer chromatogra-
phy.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902505.
Chem. Eur. J. 2010, 16, 8433 – 8438
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8433

A sensitive and selective assay for thiols is thus of great
biological importance. Much work has been done in the
area of developing fluorescent sensors for thiol detection.[7]
In almost all of the sensing systems, thiols have been as-
sayed by measuring the changes in the fluorescence signals
of the probes with the analyte concentrations in the solution
state by using a spectrofluorometer.[7] For real-world appli-
cations, however, it is preferable to perform the bioassays
on solid supports because it requires no complex and expen-
sive equipment and is thus simple, quick and convenient.[8]
To the best of our knowledge, no report, however, can be
found in the literature about thiol assays in the solid state.
A thorny problem often encountered by traditional fluo-
rophores is aggregation-caused quenching (ACQ) of their
emissions in the solid state. When dispersed in aqueous
media or bound to biomolecules, the fluorophore molecules
are inclined to aggregate, which usually quenches their fluo-
rescence processes and thus greatly limits their effective
ranges as bioprobes. The ACQ effect also makes it difficult
to assay low-abundance molec-
Results and Discussion
TPE is one of the best-known AIE fluorogens, partially due
to the great simplicity in its synthesis and derivatization.[10]
TPE and its derivatives have been widely used in the devel-
opment of bioprobe systems with high sensitivity, but selec-
tivity remains an issue of concern. It is envisioned that intro-
ducing a functional group with orthogonal reactivity to the
TPE structure will generate a TPE derivative with selectivi-
ty or specificity when it is used as an AIE-active bioprobe.
Maleimide (MI) is known for its orthogonal reactivity to
thiol in the hydrothiolation or thiol–ene click reactions
under mild conditions.[12] We thus designed the molecular
structure of an MI-functionalized TPE derivative and elabo-
rated a reaction scheme for the synthesis of the TPE-MI
adduct.
According to the synthetic route shown in Scheme 1, an
aminated TPE derivative (TPE-AM) was first prepared by
the McMurry cross-coupling reaction of 4-methylbenzophe-
ular species in biological sys-
tems because the fluorescence
signals from the miniscule
amounts of fluorophores match-
ing the bioanalyte levels may
be too weak to be determined
accurately, while at the high flu-
orophore concentrations, the
emissions are further weak-
ened, rather than enhanced,
due to the notorious ACQ
effect.
We have recently discovered
an extraordinary phenomenon
of aggregation-induced emis-
sion (AIE) that is exactly oppo-
site to the ACQ effect discussed
above.[9–11] A series of fluorogen
molecules that are nonemissive
Scheme 1. Syntheses of functionalized tetraphenylethene derivatives used in this work.
in solutions, such as tetrapheny-
lethene (TPE), are induced to
emit efficiently by aggregate formation. The unique AIE
effect has been utilized to develop new “turned on” type bi-
oprobes,[11] which enjoy much higher sensitivity than their
“turned off” counterparts. The selectivity of the AIE probes,
however, needs to be improved. Herein, we aimed to devel-
op an AIE bioprobe with high selectivity, while keeping its
superb sensitivity. In particular, we worked on the design of
a TPE-based AIE-active fluorogen and the preparation of
its solid state for thiol-specific detection in small and large
biomolecules ranging from amino acids to proteins and
living cells.
none with 4-aminobenzophenone catalyzed by a mixture of
Zn/TiCl4.[13] The amino group in TPE-AM was then trans-
formed to the maleamic acid group in TPE-BM by the reac-
tion with maleic anhydride. Finally, TPE-BM was amidated
and cyclized in the presence of sodium acetate and acetic
anhydride to give the desired product of TPE-MI in a high
yield. The clickable nature of TPE-MI was verified by its
model reaction with 1-butanethiol, which afforded the thio-
lated product of TPE-BSP in a yield as high as about 90%
after the reaction mixture had been stirred at room temper-
ature for a short while (2 h). All of the reaction intermedi-
ates and products were characterized by standard spectro-
scopic methods, from which satisfactory analysis data were
obtained (see the NMR spectra shown in Figure S1–S3 and
the numerical spectral data given in the Supporting Informa-
tion).
8434
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8433 – 8438

Biosensor with High Selectivity and Sensitivity
FULL PAPER
TPE-AM is nonemissive when it is molecularly dissolved
in solvents, such as THF, ethanol, chloroform, dichlorome-
thane, and DMSO. The photoluminescence (PL) spectrum
of a dilute solution of TPE-AM in THF is basically a flat
line parallel to the abscissa (Figure 1), manifesting the none-
Figure 2. TPE-MI (a) and TPE-BSP (b) in the solid state, c) a solution in
THF, and d) in a THF/water mixture with 95% water fraction. Photo-
graphs taken under normal laboratory lighting (a) and 365 nm UV illumi-
nation (b–d).
Figure 1. PL spectra of TPE-AM in THF/water mixtures with different
fractions of water (fw). Inset: TPE-AM in pure THF (fw =0%) and a
THF/water mixture with fw =95%; photographs taken under illumination
of a 365 nm UV light.
dergo selective electrophilic reaction with a thiol group.[12,15]
When a thiol group is added across the C=C double bond in
the MI unit, the n–p electronic conjugation is destroyed.
Thus, investigation of fluorescence behavior of the thiolated
TPE derivative, namely, TPE-BSP, would help clarify wheth-
er the MI unit is responsible for the PL quenching in the
TPE-MI system.
As can be seen from Figure 2, TPE-BSP is clearly AIE
active: the solution in THF is nonemissive but a solid
powder and aggregate suspension emit very bright sky-blue
light. The UV and PL spectra as well as the photographs
shown in Figures S6 and S7 in the Supporting Information
further substantiate the AIE nature of its light emission.
These results are exciting because they show that the emis-
sion of TPE-MI can be rejuvenated by an orthogonal click
reaction with thiol in the aggregate state, which constitutes
the foundation for the development of solid strips of fluores-
cent probes for thiol-specific detection.
TLC has been widely used as a handy tool in chemical re-
search, especially for monitoring propagation of chemical
transformation. The combination of TLC with spectrometric
methods has led to the development of a variety of conven-
ient analytical techniques.[16] We have been interested in ex-
panding the usefulness of TLC[17] and in this work, we tried
to use TLC plates as matrix strips to develop solid-state flu-
orescent probes for the detection of thiol-containing biomol-
ecules. The probe strips were prepared by dropping tiny ali-
quots of a solution of TPE-MI in dichloromethane (ꢀ5 mm)
onto the TLC plates. The plates were then dipped into solu-
tions of the bioanalytes in DMSO for 1 s. After partial evap-
oration of solvent, the TLC plates were examined under
UV illumination.
missive nature in the solution state. However, when large
amounts of water (fw >80 vol%) are added to THF, intense
emission spectra are recorded under identical measurement
conditions. Since water does not dissolve TPE-AM, the fluo-
rogenic molecules must have aggregated in the aqueous
mixtures with high water fractions. The PL data thus prove
that TPE-AM behaves just like its parent form of TPE, that
is, it is AIE active.
The aggregates of the TPE-AM molecules are nanodi-
mensional in size, as proven by the example of TEM images
shown in Figure S4 in the Supporting Information and by
the leveled-off tails in the long-wavelength region in the ab-
sorption spectra of TPE-AM in the THF/water mixtures
with high fw values (Figure S5 in the Supporting Informa-
tion). The photographs given in the inset of Figure 1 clearly
show the nonemissive and emissive nature of the molecular
species and aggregative particles of TPE-AM in the THF so-
lution and aqueous suspension, respectively. When the mole-
cules of TPE-AM are aggregated in the THF/water mixture
with 95% water, its emission intensity is increased by
around 200 fold, in comparison to that of the solution in
THF.
Whereas the nanoaggregates of TPE-AM are highly emis-
sive, the TPE-MI congener is nonfluorescent in either solu-
tion or solid state (Figure 2). The fluorescence of TPE-MI is
probably quenched by the exciton annihilation process asso-
ciated with the n–p electronic conjugation of the carbonyl
(C=O) and olefinic (C=C) groups in its MI unit.[14] If this is
the case, breaking the n–p conjugation should help to recu-
perate the AIE activity. The MI unit is known to readily un-
Before dipping the TLC plates into the bioanalyte solu-
tions, the TPE-MI spots are nonemissive under both 254
Chem. Eur. J. 2010, 16, 8433 – 8438
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8435

B. Z. Tang et al.
and 365 nm UV illuminations (Figure S8, panels a and b;
Supporting Information). After being treated by a solution
of l-cysteine, the dark spot of TPE-MI on the TLC strip
turns to bluish under the illumination of a 254 nm UV light
(Figure 3a). When excited by a 365 nm UV beam, the spot
are still clearly visible under UV illumination. Although the
spot on the TLC strip treated with the 1 ngmLÀ1 (ꢀ1 ppb)
solution of l-cysteine is somewhat weak, it is still discerna-
ble by the naked eye, demonstrating the high sensitivity or
low detection limit of the bioprobe in the solid state.[7]
An alternative way for assaying a trace amount of a bio-
analyte is to first treat a blank TLC plate with a solution of
TPE-MI and then drop an aliquot (ꢀ1 mL) of a solution of
bioanalyte onto the pretreated TLC plate. Figure S9 in the
Supporting Information shows the experimental results ob-
tained from this alternative procedure. The data are very
similar to those shown in Figure 4, once again proving the
sensitive nature of the bioprobing system.
Since the free thiol group in l-cysteine can readily react
with TPE-MI and selectively turn on the emission of its spot
on the TLC plate in the solid state at room temperature, we
examined whether the TLC plate could be used to detect
small proteins or oligopeptides containing free thiol groups
under ambient conditions, in an effort to widen the applica-
bility of the bioprobe system. We used GSH (with a se-
quence of GCE) as the bioanalyte and performed the bioas-
say by following the experimental procedure described
above. As depicted in Figure 5, the TPE-MI spot becomes
Figure 3. Selective detection of l-cysteine (C), a standard amino acid car-
rying a free thiol unit, by a TPE-MI spot on a TLC plate. Data for other
standard amino acids containing no thiol unit, including l-aspartic acid
(D), l-leucine (L), l-phenylalanine (F), l-arginine (R), l-asparagine (N),
l-methionine (M), and l-glutamic acid (E), are shown for comparison.
Photographs were taken under illuminations of a) 254 and b) 365 nm UV
light after the TLC plates had been dipped into solutions of the amino
acids (0.3 mgmLÀ1) in DMSO with and without thiols.
emits a bright blue light (Figure 3b). In sharp contrast, the
TPE-MI spots on the TLC plates treated with other amino
acids not containing free thiol groups all remain nonfluores-
cent under the 365 nm UV illumination. Evidently, TPE-MI
has readily and selectively reacted with l-cysteine to yield a
fluorescent thiolated product in the solid state at room tem-
perature, thanks to the click nature of the involved thiol–
ene hydrothiolation reaction. The detection process is fast
and the assay procedure is simple. Furthermore, the contrast
between the spots of TPE-MI and its thiolated product on
the TLC plates is very high under UV illumination, which is
vividly discernable by the naked eye.
Figure 5. Detection of protein containing free thiol groups by TPE-MI
coated on TLC plates. Photographs were taken under a) 254 and
b) 365 nm UV illumination after the TLC plates had been dipped into
solutions of proteins (0.3 mgmLÀ1) in DMSO without (GLVPA) and with
thiol unit (GCE or GSH).
Selectivity and sensitivity are the two most important pa-
rameters that decide the goodness and usefulness of a biop-
robe. It has become clear that the TPE-MI probe is highly
selective: it works only for free thiol. To determine how sen-
sitive the bioprobe was, we performed the TLC detection
experiments using solutions of l-cysteine in different con-
centrations. The results are shown in Figure 4. The TLC
emissive after the TLC plate has been dipped into a solution
of GSH with a concentration as low as about 0.3 ppm.
When treated with a protein (GLVPA) that lacks of free
thiol group, the spot of TPE-MI remains nonemissive under
UV illumination.
plate treated with
a
solution of l-cysteine with c=
While the fluorescence detections of thiol-containing
amino acids and proteins are of interest, monitoring thiol
levels in living cells is more exciting. To explore the possibil-
ity of utilizing TPE-MI as a bioprobe for cell imaging, we
tried to map the thiol levels in HeLa cells using a standard
cell-staining protocol. After incubating the living HeLa cells
in the culture buffer in the presence of the TPE-MI nanoag-
gregates for 5 min, bright fluorescence images of the stained
HeLa cells were recorded (Figure 6). Closer scrutinization
of the cell images reveals that the TPE-MI aggregates pre-
dominantly visualize the cytoplasmic regions. Weaker fluo-
rescence signals are seen in the nucleus regions of the HeLa
cells, probably due to the hydrophobic nature of the fluoro-
gen.
1000 ngmLÀ1 (ꢀ1 ppm) shows a spot with very bright fluo-
rescence. Decreasing the solution concentration from 100
and 10 ngmLÀ1 lowers the emission intensity, but the spots
Figure 4. Sensitivity in fluorescent probing of l-cysteine by TPE-MI spots
on TLC plates. Photographs were taken under 365 nm UV illumination
after the TLC plates had been dipped into solutions of l-cysteine in
DMSO with concentrations of 1–1000 ngmLÀ1
.
8436
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8433 – 8438

Biosensor with High Selectivity and Sensitivity
FULL PAPER
lective. We are currently working on the expansion of the
scope of its applications in biomolecular sensing and cellular
imaging systems.
Acknowledgements
This project was partially supported by the Research Grants Council of
Hong Kong (603008 and 602706), the University Grants committee of
Hong Kong (AoE/P-03/08), the National Science Foundation of China
(20974028), and the Ministry of Science
& Technology of China
(2009CB623605). B.Z.T. thanks the support from Cao Guangbiao Foun-
dation of Zhejiang University.
Figure 6. a) Bright field and b) fluorescent images of HeLa cells stained
by TPE-MI nanoaggregates.
[1] a) M. Sameiro T. Goncalves, Chem. Rev. 2009, 109, 190; b) I. L.
Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Nat. Mater.
2005, 4, 435.
[2] a) A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher, T. E. Rice, Chem. Rev. 1997,
97, 1515; b) W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Or-
den, J. H. Werner, R. A. Keller, Chem. Rev. 1999, 99, 2929; c) W. F.
Patton, Biotechniques 2000, 28, 944.
The cell-imagining results show that the TPE-MI nanoag-
gregates can easily penetrate the cell membranes, possibly
through an endocytosis process, and readily realize fluores-
cence labeling under the standard cell incubation conditions.
All of the living cells stained by the TPE-MI nanoaggregates
look healthy, indicative of good biocompatibility of the AIE
fluorogen. Taking into consideration the relatively high
levels of GSH in the cytosolic regions and the relationship
between the cytosolic GSH levels and many health prob-
lems,[6] this thiol-specific bioprobe may find an array of bio-
medicinal and biotech applications as a simple visualization
tool for mapping cytosolic GSH levels in the living systems.
[3] T. Terai1, T. Nagano, Curr. Opin. Chem. Biol. 2008, 12, 515.
[4] a) Z. A. Wood, E. Schrçder, J. R. Harris, L. B. Poole, Trends Bio-
chem. Sci. 2003, 28, 32; b) S. Shahrokhian, Anal. Chem. 2001, 73,
5972; c) Homocysteine in Health and Disease (Eds.: R. Carmel,
D. W. Jacobsen), Cambridge University Press, Cambridge, 2001.
[5] a) R. H. Schirmer, J. G. Mꢂller, R. L. Krauth-Siegel, Angew. Chem.
1995, 107, 153; Angew. Chem. Int. Ed. Engl. 1995, 34, 141; b) R. L.
Krauth-Siegel, H. Bauer, R. H. Schirmer, Angew. Chem. 2005, 117,
698; Angew. Chem. Int. Ed. 2005, 44, 690; c) T. P. Dalton, H. G.
Shertzer, A. Puga, Annu. Rev. Pharmacol. Toxicol. 1999, 39, 67;
d) S. M. Kanzok, R. H. Schirmer, I. Tꢂrbachova, R. Iozef, K.
Becker, J. Biol. Chem. 2000, 275, 40180; e) R. Hong, G. Han, J. M.
Fernꢃndez, B.-J. Kim, N. S. Forbes, V. M. Rotello, J. Am. Chem. Soc.
2006, 128, 1078.
Conclusion
[6] a) H. Refsum, P. M. Ueland, O. Nygard, S. E. Vollset, Annu. Rev.
Med. 1989, 40, 31; b) S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques,
I. H. Rosenberg, R. B. D’Agostino, P. W. F. Wilson, New Engl. J.
Med. 2002, 346, 476; c) D. M. Townsend, K. D. Tew, H. Tapiero,
Biomed. Pharmacother. 2003, 57, 145; d) R. Njꢄlsson, S. Norgren,
Acta Paediatr. 2005, 94, 132; e) L. A. Herzenberg, S. C. De Rosa,
J. G. Dubs, M. Roederer, M. T. Anderson, S. W. Ela, S. C. Deresin-
ski, L. A. Herzenberg, Proc. Natl. Acad. Sci. USA 1997, 94, 1967.
[7] a) H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki, N.
Itoh, Angew. Chem. 2005, 117, 2982; Angew. Chem. Int. Ed. 2005,
44, 2922; b) J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G.
Skene, J. W. Keillor, J. Am. Chem. Soc. 2007, 129, 11969; c) T. Mat-
sumoto, Y. Urano, T. Shoda, H. Kojima, T. Nagano, Org. Lett. 2007,
9, 3375; d) S. Mare, S. Penugonda, N. Ercal, Biomed. Chromatogr.
2005, 19, 80; e) A. M. Piggott, P. Karuso, Anal. Chem. 2007, 79,
8769; f) P. K. Pullela, T. Chiku, M. J. Carvan III, D. S. Sem, Anal.
Biochem. 2006, 352, 265; g) W. Jiang, Q. Fu, H. Fan, J. Ho, W.
Wang, Angew. Chem. 2007, 119, 8597; Angew. Chem. Int. Ed. 2007,
46, 8445; h) J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder, S. A.
Hilderbrand, Org. Lett. 2008, 10, 37; i) A. Shibata, K. Furukawa, H.
Abe, S. Tsuneda, Y. Ito, Bioorg. Med. Chem. Lett. 2008, 18, 2246;
j) H. Maeda, K. Katayama, H. Matsuno, T. Uno, Angew. Chem.
2006, 118, 1842; Angew. Chem. Int. Ed. 2006, 45, 1810; k) Y. H.
Ahn, J. S. Lee, Y. T. Chang, J. Am. Chem. Soc. 2007, 129, 4510; l) P.
Root, B. Mutus, Anal. Biochem. 2003, 320, 299; m) Y. Fu, H. Li, W.
Hu, D. Zhu, Chem. Commun. 2005, 3189; n) M. Zhang, M. Yu, F.
Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu, J. Zhang, D. Zhang, T. Yi,
C. Huang, J. Am. Chem. Soc. 2007, 129, 10322; o) L. Yi, H. Li, L.
Sun, L. Liu, C. Zhang, Z. Xi, Angew. Chem. 2009, 121, 4094; Angew.
Chem. Int. Ed. 2009, 48, 4034.
AIE fluorogens commonly show very high sensitivity be-
cause the emissions are from their collective assemblies,
often in the form of nanoaggregates. There is, however,
much room for improvement in terms of selectivity or spe-
cificity. Herein, an AIE bioprobe with not only high sensitiv-
ity but also high selectivity has been described that utilizes a
thiol–ene click reaction mechanism. Functionalization of
TPE by an MI group completely quenches the fluorescence
process of TPE in both solution and in the solid state. The
AIE activity, however, is recovered by the orthogonal hy-
drothiolation reaction of the MI unit with the free thiol
group in the aggregate state under ambient conditions.
This unique process has enabled the development of a
solid-state, thiol-specific bioprobe system based on TPE-MI
spots on TLC plates. Thus, thanks to the remarkable click
nature of the thiol–ene addition, the bioprobe plates selec-
tively respond to the amino acid (l-cysteine) and protein
(GSH) containing thiol groups at concentrations down to
approximately 1 ppb, but not to other small and large bio-
molecules that lack free thiol units. The TPE-MI nanoaggre-
gates show excellent performance as a visualization agent
for mapping thiol distributions in living cells under normal
cell incubation conditions. The biosensing process is simple
and convenient, in addition to being highly sensitive and se-
Chem. Eur. J. 2010, 16, 8433 – 8438
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8437

B. Z. Tang et al.
[8] a) A. Leino, B. M. Loo, Ann. Clin. Biochem. 2007, 44, 563; b) W. Pu-
talun, H. Tanaka, Y. Shoyama, Phytochem. Anal. 2005, 16, 370.
[9] a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S.
Kwok, X. Zhan, Y. Liu, D. Zhu, B. Z. Tang, Chem. Commun. 2001,
1740; b) Y. Liu, X. Tao, F. Wang, X. Dang, D. Zou, Y. Ren, M.
Jiang, J. Phys. Chem. C 2008, 112, 3975; c) B. K. An, S. K. Kwon,
S. D. Jung, S. Y. Park, J. Am. Chem. Soc. 2002, 124, 14410; d) H. C.
Yeh, S. J. Yeh, C. T. Chen, Chem. Commun. 2003, 2632; e) Z. Zhao,
Z. Wang, P. Lu, C. Y. K. Chan, D. Liu, J. W. Y. Lam, H. H. Y. Sung,
I. D. Williams, Y. Ma, B. Z. Tang, Angew. Chem. 2009, 121, 7744;
Angew. Chem. Int. Ed. 2009, 48, 7608; f) W. Z. Yuan, P. Lu, S. Chen,
J. W. Y. Lam, Z. Wang, Y. Liu, H. S. Kowk, Y. Ma, B. Z. Tang, Adv.
Mater. 2010, 22, 2159.
[10] For reviews, see: a) H. Hong, J. W. Y. Lam, B. Z. Tang, Chem.
Commun. 2009, 4332; b) J. Liu, J. W. Y. Lam, B. Z. Tang, J. Inorg.
Organomet. Polym. Mater. 2009, 19, 249; c) J. Liu, J. W. Y. Lam,
B. Z. Tang, Chem. Rev. 2009, 109, 5799; d) M. Wang, G. Zhang, D.
Zhang, D. Zhu, B. Z. Tang, J. Mater. Chem. 2010, 20, 1858.
[11] a) H. Tong, Y. Hong, Y. Dong, M. Hꢅubler, Z. Li, J. W. Y. Lam, Z.
Li, Y. Dong, H. H. Y. Sung, I. D. Williams, B. Z. Tang, J. Phys.
Chem. B 2007, 111, 11817; b) Y. Hong, M. Haussler, J. W. Y. Lam, Z.
Li, K. Sin, Y. Dong, H. Tong, J. Liu, A. Qin, R. Renneberg, B. Z.
Tang, Chem. Eur. J. 2008, 14, 6428; c) H. Tong, Y. Hong, Y. Dong,
M. Haussler, J. W. Y. Lam, Z. Li, Z. Guo, Z. Guo, B. Z. Tang, Chem.
Commun. 2006, 3705; d) L. Liu, G. Zhang, J. Xiang, D. Zhang, D.
Zhu, Org. Lett. 2008, 10, 4581; e) Y. Hong, H. Xiong, J. W. Y. Lam,
M. Hꢅubler, J. Liu, Y. Yu, Y. Zhong, H. H. Y. Sung, I. D. Williams,
K. S. Wong, B. Z. Tang, Chem. Eur. J. 2010, 16, 1232; f) M. Faisal, Y.
Hong, J. Liu, Y. Yu, J. W. Y. Lam, A. Qin, P. Lu, B. Z. Tang, Chem.
Eur. J. 2010, 16, 4266.
[12] a) R. K. Iha, K. L. Wooley, A. M. Nystrom, D. J. Burke, M. J. Kade,
C. J. Hawker, Chem. Rev. 2009, 109, 5620; b) A. Qin, J. W. Y. Lam,
B. Z. Tang, Chem. Soc. Rev. 2010, 39, DOI: 10.1039/b909064a;
c) C. K. W. Jim, A. Qin, J. W. Y. Lam, F. Mahtab, Y. Yu, B. Z. Tang,
Adv. Funct. Mater. 2010, 20, 1319; d) B. S. Sumerlin, A. P. Vogt,
Macromolecules 2010, 43, 1.
[13] a) J. E. McMurry, Chem. Rev. 1989, 89, 1513; b) B. E. Kahn, R. D.
Rieke, Chem. Rev. 1988, 88, 733; c) X. F. Duan, J. Zeng, J. W. Lu,
Z. B. Zhang, J. Org. Chem. 2006, 71, 9873.
[14] a) M. E. Langmuir, J.-R. Yang, A. M. Moussa, R. Laura, K. A. Le-
Compte, Tetrahedron Lett. 1995, 36, 3989; b) J.-R. Yang, M. E. Lang-
muir, J. Heterocycl. Chem. 1991, 28, 1177; c) S. Girouard, M. H.
Houle, A. Grandbois, J. W. Keillor, S. W. Michnick, J. Am. Chem.
Soc. 2005, 127, 559.
[15] a) T. Lee, R. L. Heintz, Eur. J. Biochem. 2001, 268, 3793; b) K. Tya-
garajan, E. Pretzer, J. E. Wiktorowicz, Electrophoresis 2003, 24,
2348; c) N. Venkatesan, B. H. Kim, Chem. Rev. 2006, 106, 3712.
[16] a) J. Sherma, Rev. Anal. Chem. 1995, 14, 75; b) G. W. Somsen, W.
Morden, I. D. Wilson, J. Chromatogr. A 1995, 703, 613.
[17] J. Chen, C. C. W. Law, J. W. Y. Lam, Y. Dong, S. M. F. Lo, I. D. Wil-
liams, D. Zhu, B. Z. Tang, Chem. Mater. 2003, 15, 1 535.
Received: September 10, 2009
Revised: March 15, 2010
Published online: June 11, 2010
8438
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8433 – 8438