A highly selective FRET-based fluorescent probe for detection of cysteine and homocysteine

Article abstract of DOI:10.1002/chem.200903121

Fluorescent probe: A turn-on FRET-based fluorescent probe for selective detection of cysteine (Cys) and homocysteine (Hey) under physiological conditions was synthesised (see figure). The probe features excellent selectivity, fast response times and good linearity towards Cys and Hey detection

Full text of DOI:10.1002/chem.200903121

DOI: 10.1002/chem.200903121
A Highly Selective FRET-Based ACTHNUGRTENUNGFluorescent Probe for Detection of Cysteine
and Homocysteine
Hoi-Yan Shiu,[a] Hiu-Chi Chong,[b] Yun-Chung Leung,[b] Man-Kin Wong,*[b] and
Chi-Ming Che*[a]
Biological thiols including cysteine (Cys), homocysteine
(Hcy) and glutathione (GSH) play significant roles in redox-
related biological processes.[1] Abnormal levels of Cys and
Hcy are associated with a variety of human diseases such as
slow growth,[2] liver damage,[2] skin lesions,[2] Alzheimerꢀs
diseases,[3a] cardiovascular diseases[3a,b] and coronary heart
diseases.[3c] Thus, the development of efficient methods for
the detection and quantification of biological thiols under
physiological conditions is of importance in both chemistry
and medicine.
Analytical methods for the detection of thiols in biologi-
cal samples have been substantially advanced owing to the
development of analytical techniques including high-perfor-
mance liquid chromatography (HPLC),[4] capillary electro-
phoresis (CE),[5] electrochemical assay[6] and mass spectrom-
etry.[7] However, laborious procedures such as sample clean-
up, isolation and purification are often involved before in-
strumental analysis can take place. Thus, there has been a
surge of interest in the development of simple and yet sensi-
tive thiol detection methods in which the requirement of te-
dious sample preparation procedures and expensive instru-
mentation can be minimised.
namic range. In addition, inexpensive instrumentation and
simple procedures are involved.[8] The pioneering work by
Strongin and co-workers on the use of an aldehyde-contain-
ing xanthene dye for fluorescent detection of Cys or Hcy[9]
has stimulated considerable interest to develop new fluores-
cent probes for biological thiol detection.[10] However, limi-
tations exist for the reported fluorescent thiol probes, in-
cluding high background fluorescence[11] and long response
times.[12] Therefore, it remains a challenging task to develop
highly selective and efficient fluorescent probes for the de-
tection of Cys and Hcy.
Along with our long-term interest in the design and syn-
thesis of novel luminescent materials for solving biological
problems, we recently explored the use of luminescent
metal complexes for protein staining and cell imaging.[13] In
the search for new bioconjugation reactions,[14a] we have re-
cently developed a method for selective and reversible
modification of cysteine-containing peptides by electron-de-
ficient alkynones in aqueous medium (Scheme 1).[14b]
As depicted in Scheme 1, the thiol group of cysteine-con-
taining peptides can be linked to electron-deficient alkyn-
AHCTUNGTREGoNNNU nes to generate a vinyl sulfide linkage. Upon treatment
Fluorescent molecular probes are advantageous because
fluorescence detection is highly sensitive and has a good dy-
with excess thiol (aromatic or aliphatic), an intermediate di-
thioacetal is formed. The vinyl sulfide linkage of the dithio-
AHCTUNGTREGaNNUN cetal can be cleaved to regenerate free thiol through elimi-
nation of molecule A. Note that the vinyl sulfide linkage
can only be cleaved by addition of thiols but not other nu-
cleophiles such as amines and alcohols. We envision that
this thiol-specific cleavage reaction could be employed to
develop a highly selective probe for the detection of thiols.
An ideal fluorescent probe should be triggered to give a
strong fluorescent signal upon selective reaction with its
target analytes. With the thiol-specific cleavage reaction in
hand, we set out to design and synthesise a “turn-on” fluo-
rescent probe for thiol detection. We proposed to connect a
fluorophore and a quencher through the vinyl sulfide link-
age. The resulting fluorescent probe should be weakly fluo-
rescent (“turn-off”) owing to quenching through fluores-
cence resonance energy transfer (FRET).[15] Upon treatment
[a] H.-Y. Shiu, Prof. Dr. C.-M. Che
Department of Chemistry and Open Laboratory of
Chemical Biology of the Institute of Molecular Technology
for Drug Discovery and Synthesis, The University of Hong Kong
Pokfulam Road, Hong Kong (China)
Fax : (+852)2857-1586
E-mail: cmche@hku.hk
[b] H.-C. Chong, Dr. Y.-C. Leung, Dr. M.-K. Wong
Department of Applied Biology and Chemical Technology
The Hong Kong Polytechnic University
Hung Hom, Kowloon, Hong Kong (China)
Fax : (+852)2364-9932
E-mail: bcmkwong@inet.polyu.edu.hk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903121.
3308
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3308 – 3313

COMMUNICATION
mation). Fluorescein absorbs
at 480 nm and emits at 515 nm
(green-coloured
fluorescen-
ce),[15a] whereas the diarylazo
quencher exhibits maximum
light absorption at 515 nm.[17]
Therefore, probe 1 is non-fluo-
rescent because there is signifi-
cant spectral overlap between
Scheme 1. Electron-deficient alkynones as cleavable reagents for modification of cysteine-containing peptides
in aqueous medium.
fluorescein
emission
and
quencher absorption spectra,
with thiols, the quencher unit is disconnected from the
probe through cleavage of the vinyl sulfide linkage. As a
result, the fluorescent probe is turned-on to give a strong
fluorescent signal (Scheme 2).
The turn-on mechanism of the FRET-based fluorescent
probe was designed on the basis of a highly selective conju-
gate 1,4-addition of thiols to an a,b-unsaturated vinyl sulfide
linkage followed by elimina-
that is, excitation at 480 nm, and results in limited emission
at 515 nm. Because FRET is distance dependent (effective
within 100 ꢂ), cleavage of 1 by thiols separates the fluores-
cein from the quencher. Therefore, the quencher (1Q)
could no longer effectively quench the emission from the
fluorophore (1F). As a result, a strong emission at 515 nm
from 1F was detected.
tion. We envision that the rate
of this conjugate 1,4-addition
is dependent on the steric bulk
of thiol nucleophiles. It is ex-
pected that this fluorescent
probe is able to differentiate
thiols of different steric bulk.
Hcy, which has the least bulky
thiol group, would give higher
fluorescent signal than Cys,
whereas no significant signal
would be obtained for sterical-
ly bulky GSH. A ketocoumar-
in-based thiol detection probe
featuring conjugate 1,4-addi-
tion of thiols to a,b-unsaturat-
ed ketones based on an intra-
molecular charge system (ICT)
has recently been reported.[16]
However, the reported probe
displays little selectivity among
different thiol-containing com-
pounds. Herein, we report the
development of a highly selec-
tive turn-on fluorescent probe
1 for detection of Cys and Hcy
Scheme 2. Turn-on fluorescence can be achieved after removal of the quencher by the thiol-assisted cleavage
of the vinyl sulfide linkage.
by connecting
a
diarylazo
quencher and a fluorescein flu-
orophore through the vinyl
sulfide linkage.[17]
Scheme 3 depicts the work-
ing principle of 1 as a fluores-
cent sensor for Cys and Hcy.
Probe 1 was prepared by con-
necting a diarylazo quencher
and a fluorescein fluorophore
through the vinyl sulfide link-
age (see the Supporting Infor-
Scheme 3. Working principle of 1 as a fluorescent sensor in response to cysteine and homocysteine.
Chem. Eur. J. 2010, 16, 3308 – 3313
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3309

C.-M. Che, M.-K. Wong et al.
At first, the time course of the reaction between probe 1
and Hcy was studied by monitoring the fluorescence intensi-
ty of the reaction mixture at 515 nm (Figure 1). A solution
of 1 (5 mm) in phosphate-buffered saline (PBS buffer,
Figure 2. A photograph showing the fluorescence emission of 1 when
treated with Cys or Hcy.
ESIMS analysis of the reaction mixture containing 1
(5 mm) and Hcy (0.2 mm) revealed three signals. In addition
to the signal from unreacted 1 (m/z=1106.4), the other two
signals (m/z=532.2 and 709.2) are consistent with the for-
mulation of the quencher part 1Q and the fluorescein part
1FHcy, respectively. Similarly, three signals were found in the
ESIMS analysis of the reaction mixture of 1 (5 mm) and Cys
(0.2 mm). The three species were unreacted 1 (m/z=1106.4),
the detached quencher 1Q (m/z=532.2) and the fluorescein
part 1FCys (m/z=695.2).
To study the fluorescent properties of the fluorescent
parts (1FHcy and 1FCys), a model compound 1Fmodel was con-
structed (Scheme 4, see the Supporting Information). 1Fmodel
was found to be strongly fluorescent, with FF =0.74 in
NaOH (0.1m; see the Supporting Information). To demon-
strate the linearity of the response of 1 towards Hcy, 1
(5 mm) was treated with Hcy at different concentrations in
pH 8.1 PBS buffer/DMSO (9:1), the corresponding emission
intensity at 515 nm was measured after 15 min. When the
emission intensity of 1 at 515 nm was plotted against Hcy
concentration, a calibration curve (Figure 3) revealing a
good linear relationship (R value=0.99) in the Hcy concen-
tration range of 0–0.3 mm was obtained. Moreover, the addi-
tion of Hcy to the solution resulted in up to a 40-fold in-
crease in emission intensity at 515 nm when the concentra-
tion ratio of [Hcy]/[1] increased from 0 to 60.
Figure 1. Respective time course of the reaction between 1 and homocys-
teine and cysteine in a molar ratio of 1:40.
pH 8.1) is weakly emissive (FF =0.03 in NaOH (0.1m), see
the Supporting Information). The low background fluores-
cence reveals that fluorescein is effectively quenched by the
diarylazo quencher. Upon treatment with Hcy (0.2 mm) in
pH 8.1 PBS buffer/DMSO (9:1) at room temperature, a
more than 20-fold increase in the fluorescence intensity at
515 nm was detected within the first 5 min. This abrupt in-
crease in the emission intensity signified a high initial reac-
tion rate in the cleavage of the vinyl sulfide linkage of 1 by
the nucleophilic Hcy. After 15 min the increase in emission
intensity levelled off. Therefore, an assay time of 15 min was
selected in the evaluation of the selectivity and sensitivity of
1 towards detection of thiols in subsequent studies. Up to a
30-fold increase in the fluorescence intensity was observed
when 1 (5 mm) was treated with Hcy (0.2 mm) in a molar
ratio of 1:40. Up to a 20-fold increase in the fluorescence in-
tensity was observed when 1 (5 mm) was treated with Cys
(0.2 mm) in a molar ratio of 1:40. Figure 2 shows the fluores-
cence emission of 1 when treated with Hcy or Cys.
Up to a 30-fold increase in emission intensity at 515 nm
was obtained when the concentration ratio of [Cys]/[1] in-
Scheme 4. Structures of the fluorescent parts 1FHcy, 1FCys and 1FModel
.
3310
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3308 – 3313

A Highly Selective FRET-Based Fluorescent Probe
COMMUNICATION
To examine the selectivity of the FRET-based probe, 1
was treated with various biologically relevant analytes in-
cluding amino acids, glucose, metal ions, a reactive oxygen
species and a reducing agent. Amino acids (Ala, Arg, Asn,
Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr and Val), metal ions (Na+, K+, Ca2+, Mg2+
,
Fe3+ and Zn2+), a reducing agent (nicotinamide adenine di-
nucleotide (NADH)) and glucose did not result in any emis-
sion enhancement, even at 40:1 molar ratio of the analyte
versus 1. The results demonstrated that 1 is selective to thiol
species among the various biologically relevant analytes.
H2O2 (a strong oxidising agent) gave a weak fluorescent
signal upon reaction with 1. A possible explanation may be
due to oxidative degradation of the diarylazo quencher[19]
(Figure 5)
In addition, we applied probe 1 to fluorescent sensing of
Cys/Hcy in commercial human blood plasma. The conver-
sion of disulfides to free thiols was conducted by treatment
of the plasma sample with a reducing agent, triphenylphos-
phine, according to a literature procedure.[9b] Aliquots of
commercial human blood plasma (0, 50, 70, 100, 150 or
200 mL) were added to a solution of probe 1 (5 mm) in PBS
Figure 3. Emission spectral traces of 1 (5 mm) in PBS buffer with increas-
ing [Hcy]/[1] ratio (from 0 to 60) at room temperature. Each spectrum
was acquired 15 min after Hcy addition.
creased from 0 to 60. Yet, treatment of up to 40 equivalents
of GSH (a cysteine-containing peptide bearing three amino
acids) with 1 did not result in emission enhancement. These
results indicated that 1 is responsive to the steric hindrance
of the thiol species. The intensity of the emission signal is in
the order Hcy>Cys@GSH. This is consistent with the dif-
ference in reaction rate of the thiols with different steric
bulk towards conjugate 1,4-addition.[18] Note that probe 1 is
able to differentiate between the subtle steric hindrance dif-
ference between Hcy and Cys (Figure 4). As expected,
probe 1 gave no response to sterically bulky cysteine-con-
taining biomolecules including peptide STSSSCNLSK (SK-
10), bovine serum albumin (BSA) and human serum albu-
min (HSA; Figure 4). This is of importance for probe 1 to
selectively detect Cys and Hcy in a complex biological
matrix.
Figure 4. Enhancement of emission intensity of 1 by SK-10, BSA, HSA,
GSH, Cys and Hcy.
Figure 5. Enhancement of emission intensity of 1 by different analytes.
Chem. Eur. J. 2010, 16, 3308 – 3313
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3311

C.-M. Che, M.-K. Wong et al.
buffer solution (pH 8.1) at room temperature. As shown in
Figure 6, an increase in the volume of the reduced blood
plasma leads to a corresponding linear enhancement in the
fluorescence intensity, revealing that the probe is able to
detect thiols in the plasma sample.
Figure 8. Fluorescence and brightfield images of fixed SK-HEP-1 cells:
a) fluorescence image of cells incubated with 1 (10 mm) for 10 min at
room temperature; b) brightfield image of cells shown in panel a; c) the
overlay of panels a and b.
compound) for 2h (see the Supporting Information), reveal-
ing the specificity of probe 1 towards Cys/Hcy.
In summary, a turn-on fluorescent probe 1 for the detec-
tion of thiols on the basis of conjugate addition of thiols to
vinyl sulfide linkages followed by elimination was synthes-
ised. Probe 1 features high sensitivity and excellent selectivi-
ty for Cys/Hcy over other amino acids, GSH, BSA and HSA
under physiological conditions. In addition, the probe also
features fast signal response times and a good linearity
range for quantification. We have demonstrated that 1 can
be used as an effective sensing probe for Cys/Hcy in human
blood plasma and in fixed cells by confocal laser scanning
microscopy.
Figure 6. Calibration curve obtained by treating 1 (5 mm) with different
amounts of commercial human blood plasma (0, 50, 70, 100, 150 or
200 mL).
For quantitative measurement,
a standard addition
method with Hcy as the standard was employed to estimate
the unknown concentration of thiols in the commercial
human blood plasma. The total content of thiols in the
plasma was found to be 0.31 mm (see the Supporting Infor-
mation), which is well within the range of reported thiol
concentrations from normal human blood plasma.[20]
We have employed probe 1 to detect Cys and Hcy in cells
(MKN-45 and SK-HEP-1 cells) by using confocal laser scan-
ning microscopy. As shown in Figures 7a and 8a, a signifi-
cant fluorescence enhancement was observed after incubat-
ing the fixed cells with a solution of 1 (10 mm) in ethanol/
PBS (7:3 v/v, pH 7.4) for 10 min at 258C. The brightfield
images of MKN-45 and SK-HEP-1 cells were also recorded.
The overlay of fluorescence and brightfield images (Figur-
es 7c and 8c) revealed that fluorescence signals with differ-
ent intensities were localised in different compartments of
the cells. As a control, no fluorescence was observed for
cells pre-treated with N-methylmaleimide (a thiol-reactive
Experimental Section
General: Chemicals purchased from commercial sources were used with-
out further purification. Electronic absorption spectra were recorded
with a HP Agilent 8453 UV/Vis spectrophotometer. The emission spectra
were measured on a PerkinElmer LS 55 fluorescence spectrometer. TLC
analyses were performed on silica-gel plates and flash column chroma-
tography was conducted over silica gel 60 (230–400 mesh ASTM) with
ethyl acetate/n-hexane or methanol/dichloromethane as the eluent. 1H
and 13C NMR spectra were recorded on a Bruker DPX-300 or DPX-400
spectrometer. Chemical shifts (ppm) are referenced to TMS. Mass spec-
tra were measured by using
a hybrid QTOF mass spectrometer
(QSTAR-XL system, ABI, USA) and Finnigan MAT 95 or LCQ mass
spectrometer.
Spectral measurements: The amino acids (Cys, Hcy, Ala, Arg, Asn, Asp,
Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and
Val), GSH, metal ions (Na+, K+, Ca2+, Mg2+, Fe3+ and Zn2+), reactive
oxygen species (hydrogen peroxide), reducing agent (NADH), glucose,
peptide STSSSCNLSK (SK-10), BSA and HSA stock solutions were pre-
pared in doubly distilled water. Probe 1 was dissolved in DMSO at room
temperature to afford the probe stock solution (0.1 mm). The probe stock
solution (20 mL) and the analyte stock solution (8 mL) were added to a
solvent mixture of DMSO (20 mL) and PBS buffer (pH 8.1, 352 mL). The
resulting solution was shaken well. After 15 min, the emission spectra
were recorded. Unless otherwise stated, for all measurements the excita-
tion wavelength was 480 nm and the excitation and emission slit widths
were 5 nm. The thiol assay in human blood plasma typically requires the
reduction of disulfides to free thiols. This can be accomplished by using
an excess of triphenylphosphine at room temperature for 30 min. Ali-
quots of the human blood plasma after reduction were then directly
added to probe 1 (5 mm) and diluted to 400 mL with pH 8.1 PBS buffer so-
lution, and the emission at 515 nm was recorded. The unknown amount
of thiols in human blood plasma sample was estimated by using the stan-
dard addition method with Hcy as the standard.
Figure 7. Fluorescence and brightfield images of fixed MKN-45 cells:
a) fluorescence image of cells incubated with 1 (10 mm) for 10 min at
room temperature; b) brightfield image of cells shown in panel a; c) the
overlay of panels a and b.
3312
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3308 – 3313

A Highly Selective FRET-Based Fluorescent Probe
COMMUNICATION
Cell culture: MKN-45 and SK-HEP-1 cells were maintained in DMEM
(Dulbeccoꢀs modified eagleꢀs medium) and MEM (minimum essential
medium), respectively, which were both supplemented with fetal bovine
serum (10% v/v) and penicillin/streptomycin (100 unitsmLÀ1). The cells
(1ꢃ104) were seeded to the eight-well chamber slide (9ꢃ9 mm each) and
allowed to adhere for 24 h at 378C, 95% air and 5% CO2.
son, F. H. Strobel, M. Reed, J. Pohl, D. P. Jones, Clin. Chim. Acta
2008, 396, 43–48.
[8] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed.,
Plenum Publishing, New York, 1999.
[9] a) O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, J. Am. Chem.
Soc. 2004, 126, 438–439; b) W. Wang, J. O. Escobedo, C. M. Law-
rence, R. M. Strongin, J. Am. Chem. Soc. 2004, 126, 3400–3401;
c) W. Wang, O. Rusin, X. Xu, K. K. Kim, J. O. Escobedo, S. O. Fa-
kayode, K. A. Fletcher, M. Lowry, C. M. Schowalter, C. M. Law-
rence, F. R. Fronczek, I. M. Warner, R. M. Strongin, J. Am. Chem.
Soc. 2005, 127, 15949–15958; d) D. Wang, W. E. Crowe, R. M.
Strongin, M. Sibrian-Vazquez, Chem. Commun. 2009, 1876–1878.
[10] a) F. Tanaka, N. Mase, C. F. Barbas III, Chem. Commun. 2004,
1762–1763; b) S. Sreejith, K. P. Divya, A. Ajayaghosh, Angew.
Chem. 2008, 120, 8001–8005; Angew. Chem. Int. Ed 2008, 47, 7883–
7887; c) J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder, S. A. Hil-
derbrand, Org. Lett. 2008, 10, 37–40; d) K.-S. Lee, T.-K. Kim, J. H.
Lee, H.-J. Kim, J.-I. Hong, Chem. Commun. 2008, 6173–6175; e) Y.
Kanaoka, M. Machida, K. Ando, T. Sekine, Biochim. Biophys. Acta
1970, 207, 269–277; f) T. O. Sippel, J. Histochem. Cytochem. 1981,
29, 314–316; g) K. Yamamoto, T. Sekine, Y. Kanaoka, Anal. Bio-
chem. 1977, 79, 83–94.
Fluorescence imaging: Confocal fluorescence imaging was performed
with a Zeiss LSM510 META laser scanning microscope with a 40ꢃ ob-
jective lens. Green fluorescence was excited at 488 nm with an argon
laser and emission was collected by a 515–565 nm band pass filter. The
differential interference contrast (DIC) and the fluorescence was cap-
tured, digitised and processed to generate the pseudo-colour images by
using Zeiss LSM Image Examiner software. For fixed cell imaging, imme-
diately before the experiments cells were washed with the PBS buffer
and then incubated with 1 (10 mm) in ethanol/PBS solution (7:3 v/v,
pH 7.4) for 10 min at 258C. Fluorescence imaging was then carried out
after washing the cells with the PBS buffer. For the control experiment,
the cells were pre-treated with a solution of N-methylmaleimide (50 mm)
in an ethanol/PBS solution (70:30, v/v, pH 7.4) for 2 h in the incubator,
and then incubated with 1 (10 mm) in an ethanol/PBS solution (70:30 v/v,
pH 7.4) for 10 min at 258C.
[11] L. Duan, Y. Xu, X. Qian, F. Wang, J. Liu, T. Cheng, Tetrahedron
Lett. 2008, 49, 6624–6627. A near IR dye for the ratiometric thiol
quantification mentioned in reference [10b] can address to the prob-
lem of high background fluorescence.
Acknowledgements
[12] 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,
10322–10323. We have synthesized the reported probe according to
the method mentioned therein. Maximum fluorescence enhance-
ment (75-fold increase) was attained after treating the probe with
Hcy (40 equiv) for 80 min.
[13] a) P. Wu, E. L.-M. Wong, D.-L. Ma, G. S.-M. Tong, K.-M. Ng, C.-M.
Che, Chem. Eur. J. 2009, 15, 3652–3656; b) D.-L. Ma, C.-M. Che, S.-
C. Yan, J. Am. Chem. Soc. 2009, 131, 1835–1846; c) P. K.-M. Siu, D.-
L. Ma, C.-M. Che, Chem. Commun. 2005, 1025–1027; d) D.-L. Ma,
C.-M. Che, Chem. Eur. J. 2003, 9, 6133–6144; e) C.-M. Che, J.-L.
Zhang, L.-R. Lin, Chem. Commun. 2002, 2556–2557; f) H.-Q. Liu,
S.-M. Peng, C.-M. Che, J. Chem. Soc. Chem. Commun. 1995, 509–
510.
We are grateful for the financial support of The University of Hong
Kong (University Development Fund), Hong Kong Research Grants
Council (PolyU 7052/07P), The Hong Kong Polytechnic University
(PolyU Departmental General Research Funds and Competitive Re-
search Grants for Newly Recruited Junior Academic Staff) and the
Areas of Excellence Scheme established under the University Grants
Committee of the Hong Kong Special Administrative Region, China
(AoE/P-10/01).
Keywords: bioimaging · fluorescence · FRET · quenchers ·
thiols
[14] a) W.-K. Chan, C.-M. Ho, M.-K. Wong, C.-M. Che, J. Am. Chem.
Soc. 2006, 128, 14796–14797; b) H.-Y. Shiu, T.-C. Chan, C.-M. Ho,
Y. Liu, M.-K. Wong, C.-M. Che, Chem. Eur. J. 2009, 15, 3839–3850.
[15] For review on FRET, see: a) K. E. Sapsford, L. Berti, I. L. Medintz,
Angew. Chem. 2006, 118, 4676–4704; Angew. Chem. Int. Ed. 2006,
45, 4562–4588. For selected examples that use FRET, see: b) M. J.
Hangauer, C. R. Bertozzi, Angew. Chem. 2008, 120, 2428–2431;
Angew. Chem. Int. Ed. 2008, 47, 2394–2397; c) reference [8].
[16] W. Lin, L. Yuan, Z. Cao, Y. Feng, L. Long, Chem. Eur. J. 2009, 15,
5096–5103.
[1] a) R. Hong, G. Han, J. M. Fernꢄndez, B.-J. Kim, N. S. Forbes, V. M.
Rotello, J. Am. Chem. Soc. 2006, 128, 1078–1079; b) N. F. Zakharch-
uk, N. S. Borisova, T. V. Titova, J. Anal. Chem. 2008, 63, 171–179;
c) M. Kemp, Y.-M. Go, D. P. Jones, Free Radical Biol. Med. 2008, 44,
921–937.
[2] S. Shahrokhian, Anal. Chem. 2001, 73, 5972–5978.
[3] a) S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. Dꢀ Agostino, P. W. F. Wilson, P. A. Wolf, N. Engl. J. Med. 2002,
346, 476–483; b) H. Refsum, P. M. Ueland, O. Nygꢅrd, S. E. Vollset,
Annu. Rev. Med. 1998, 49, 31–62; c) O. Nekrassova, N. S. Lawrence,
R. G. Compton, Talanta 2003, 60, 1085–1095.
[17] A. Laikhter, M. A. Behlke, J. Walder, K. W. Roberts, Y. Yong, 2008,
US 7439341 B2.
[4] For selected examples, see: a) A. R. Ivanov, I. V. Nazimov, L. A.
Baratova, J. Chromatogr. A 2000, 870, 433–442; b) Y. Ogasawara, Y.
Mukai, T. Togawa, T. Suzuki, S. Tanabe, K. Ishii, J. Chromatogr. B
2007, 845, 157–163.
[5] For selected examples, see: a) A. R. Ivanov, I. V. Nazimov, L. A.
Baratova, J. Chromatogr. A 2000, 895, 167–171; b) T. Inoue, J. R.
Kirchhoff, Anal. Chem. 2002, 74, 1349–1354.
[6] For selected examples, see: a) D. Potesil, J. Petrlova, V. Adam, J.
Vacek, B. Klejdus, J. Zehnalek, L. Trnkova, L. Havel, R. Kizek, J.
Chromatogr. A 2005, 1084, 134–144; b) P. R. Lima, W. J. R. Santos,
R. de C. S. Luz, F. S. Damos, A. B. Oliveria, M. O. F. Goulart, L. T.
Kubota, J. Electroanal. Chem. 2008, 612, 87–96.
[18] a) A. K. Majors, S. Sengupta, B. Willard, M. T. Kinter, R. E. Pyeritz,
D. W. Jacobsen, Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1354–
1359; b) M. Gabaldon, Arch. Biochem. Biophys. 2004, 431, 178–188;
c) F. Stern, Y. N. Berner, Z. Polyak, M. Komarnitsky, B.-A. Sela, M.
Hopp, Y. Dror, Clin. Biochem. 2004, 37, 1002–1009; d) J. S. Stamler,
A. Slivka, Nutr. Rev. 1996, 54, 1–30.
[19] C. T. Fragoso, R. Battisti, C. Miranda, P. C. de Jesus, J. Mol. Catal. A
2009, 301, 93–97.
[20] Healthy plasma total homocysteine concentration is approximately
12 mm. Cysteine concentration is 20–30 times that of homocysteine
(i.e., 0.24–0.36 mm; see references [3a], [3b], [9a] and [9b]).
[7] For selected examples, see: a) N. Burford, M. D. Eelman, D. E.
Mahony, M. Morash, Chem. Commun. 2003, 146–147; b) J. M. John-
Received: November 13, 2009
Published online: February 16, 2010
Chem. Eur. J. 2010, 16, 3308 – 3313
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3313