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Interdisciplinary detection science

www.rsc.org/analyst Volume 135 | Number 1 | January 2010 | Pages 1–196

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ISSN 0003-2654
HOT ARTICLE
MINIREVIEW COMMUNICATION Wei-Lung Tseng et al.
Qian Wang et al. Andrew Mills et al. Fluorescent sensing of
Viruses and their potential in A novel reversible relative-humidity homocysteine in urine: using
bioimaging and biosensing indicator ink based on methylene fluorosurfactant-capped gold
applications blue and urea nanoparticles and o-phthaldialdehyde
 PAPER www.rsc.org/analyst | Analyst
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Fluorescent sensing of homocysteine in urine: Using fluorosurfactant-capped

gold nanoparticles and o-Phthaldialdehyde†
Jia-Hui Lin,a Chung-Wei Changa and Wei-Lung Tseng*ab
Received 11th August 2009, Accepted 21st October 2009
First published as an Advance Article on the web 2nd November 2009
DOI: 10.1039/b916511h

This study reports the development of a simple, sensitive, and selective-detection system for
homocysteine (HCys) based on the combination of fluorosurfactant-capped gold nanoparticles
(FSN-AuNPs) and o-Phthaldialdehyde (OPA). The proposed assay utilizes FSN-AuNPs as extractors
for HCys and cysteine (Cys), which can then be collected by centrifugation. As long as the HCys and
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Cys are isolated from the initial sample, they can be liberated from the NP surface by
2-mercaptoethanol (2-ME). The derivatization of released HCys with OPA/2-ME has a strong
fluorescence maximum at 485 nm, whereas the derivatization of released Cys with the same reagent
shows an extremely weak fluorescence maximum at 457 nm. As a result, the selectivity of this system is
more than 100-fold for HCys over any aminothiols when excited at 370 nm. The extraction and
derivation efficiencies are monitored as functions of the concentration of FSN-AuNPs and OPA,
respectively. The proposed system has a detection limit of 180 nM at a signal-to-noise ratio of 3 for
HCys. This study validates the applicability of this system by analyzing the amount of HCys in
urine samples.

Introduction of sensing HCys or Cys based on a ring formation of thiazinane or

HCys is a high risk factor for Alzheimer’s disease1 and cardio-
vascular disease.2 As such, the analysis of homocysteine (HCys)
levels in plasma and urine is of continuing interest to researchers.
In humans, most of the HCys synthesized each day is converted
to cysteine (Cys) and methionine under the control of a specific
enzyme. Normal concentrations of HCys in plasma and urine
range from 6.7 to 14.5 mM and 1.4 to 0.6 mol mmol1 creatinine,
respectively.3 A plasma HCys concentration greater than 15 mM
is termed as hyperhomocysteinemia, which has been classified as
moderate (15–30 mM), intermediate (30–100 mM), and severe
(> 100 mM).4 A high level of HCys can cause a variety of adverse
health effects such as neural tube defects,5 osteoporosis,6 and
cancer.7 Thus, clinical diagnosis requires the routine analysis of
HCys in biological fluid.
Current methods for determining HCys levels include gas
chromatography,8 high performance liquid chromatography,9 and
capillary electrophoresis.10 These separation techniques are
commonly coupled with laser-induced fluorescence, mass spec-
trometry, or electrochemical detection. However, all of these
methods are rather complicated, time-consuming, and costly. To
overcome these problems, researchers have proposed numerous
chemosensors for the simple, rapid, and sensitive detection of
HCys.11–14 A series of dyes containing the aldehyde group is capable
a
Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai
Road, Kaohsiung, 804, Taiwan. E-mail: tsengwl@mail.nsysu.edu.tw; Fax:
+886 7-3684046
b
National Sun Yat-sen University-Kaohsiung Medical University Joint
Research Center, Kaohsiung, Taiwan
† Electronic supplementary information (ESI) available: Fluorescence
spectrum, electrophoregram, and optimal derivatization conditions. See
DOI: 10.1039/b916511h
thiazolidine.11 Adduct formation results in fluorescence quenching
or enhancement and color change. For example, researchers have
used a xanthene dye containing the aldehyde group to recognize Cys
or HCys based on fluorescence quenching.11b Following a similar
strategy, selective fluorescent recognition of HCys has been
accomplished by selective reaction of the aldehyde group of an
iridium complex with HCys.12 Strongin’s group introduced dication
methyl viologen for sensing HCys in an organic/aqueous solution.13
A selective fluorescence turn-on sensor for the intracellular imaging
of Cys and HCys has also been accomplished using a highly elec-
tron-deficient system.14 However, the methods mentioned above
suffer from sophisticated synthesis, poor aqueous solubility, cross-
sensitivity toward Cys, and/or poor sensitivity.
Recently, researchers have used gold nanoparticles (AuNPs)
as an alternative for the detection of aminothiols.15 When ami-
nothiols attach to the AuNPs via the formation of Au–S bonds,
the AuNPs become aggregated. This is mainly because of
electrostatic interaction and hydrogen bonding between amino-
thiol-capped AuNPs. The aggregated AuNPs lead to a decrease
in surface plasmon resonance (SPR) peak at around 520 nm and
the formation of a long wavelength band (630 nm). As a result,
aminothiols can be quantified by monitoring the extinction ratio
between dispersed and aggregated NPs. Moreover, AuNPs
provide a tremendously high extinction coefficient, over
1000 times higher than that of organic dyes.16 The fluorescence of
organic dyes adsorbed on the surface of the AuNPs is severely
quenched by energy-transfer and/or charge-transfer processes.17
The presence of aminothiols removes the organic dyes from the
NP surface via the formation of Au–S bonds. As a result,
the organic dyes restore their fluorescence.15b,15c Although the
methods above have high aminothiol sensitivity, they are not
selective with respect to specific aminothiols. To achieve better

104 | Analyst, 2010, 135, 104–110 This journal is ª The Royal Society of Chemistry 2010

selectivity, researchers have used citrate-capped AuNPs modified
with nonionic fluorosurfactant (Zonyl FSN) for selective detec-
tion of Cys and HCys.18 The sensing of Cys can be successfully
achieved using FSN-capped AuNPs (FSN-AuNPs) after
aminothiols have been pretreated with NaOH.19a This may be
due to the fact that HCys forms a five-membered ring under the
pretreatment of NaOH, thereby decreasing the rate of the
aggregation of FSN-AuNPs. Moreover, when the particle size of
FSN-AuNPs changes from 12 to 40 nm, the NP aggregation
induced by HCys is faster than that induced by Cys.19b Thus,
under optimum reaction time, 40-nm FSN-AuNPs enable the
selective detection of HCys. However, using 40-nm FSN-AuNPs
as an HCys sensor reveals a narrow linear range (0.2–2.5 mM) for
the quantification of HCys because the molar ratio of HCys to
Cys determines the aggregation of 40-nm FSN-AuNPs.
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This study presents a technique for highly selective and
sensitive detection of HCys using FSN-AuNPs as an extracting
agent and o-Phthaldialdehyde (OPA) as a derivatizing agent.
FSN-AuNPs are capable of selectively extracting HCys and Cys
from an aqueous solution. 2-Mercaptoethanol (2-ME) removes
HCys and Cys from the surface of the AuNPs through ligand
exchange.20 Compared to HCys, the derivatization of Cys with
OPA/2-ME suffers from a low reaction rate21 and low fluores-
cence quantum yield.22 Moreover, the excitation and emission
wavelengths of OPA/2-ME-derivatized HCys center at 370 and
485 nm, respectively, which are entirely different from the
excitation and emission maxima of OPA/2-ME-derivatized Cys
centered at 340 and 457 nm. Based on these results, we assume
that OPA/2-ME could be used to selectively detect HCys in
a mixture of HCys and Cys, which has been extracted by
FSN-AuNPs. All important parameters were carefully evalu-
ated, including the pH of the solutions and the concentration of
FSN-AuNPs, on the sensing of HCys. To demonstrate the
practicality of this approach, this study uses it to determine the
total HCys in a urine sample.
Experimental procedures
Chemicals
HCys, Cys, glutathione (GSH), g-glutamylcysteine (Glu-Cys),
cysteinylglycine (Cys-Gly), histidine, 2-ME, trisodium citrate,
hydrogen tetrachloroaurate(III) dehydrate, sodium hydroxide,
OPA, FSN, H3PO4, NaH2PO4, Na2HPO4, Na3PO4, poly-
(diallydimethylammonium chloride) (PDDAC; 20 wt% in
water; MW 400 000–500 000), and tris(2-carboxyethyl)phosphine
(TCEP) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The molecular formula of FSN is F(CF2CF2)3–8CH2CH2O(CH2-
CH2O)xH. Water used in all experiments was doubly distilled and
purified by a Milli-Q system (Millipore, Milford, MA, USA).
Apparatus
The extinction spectra of the FSN-AuNPs were recorded using
a double-beam UV-vis spectrophotometer (Cintra 10e; GBC,
Victoria, Australia). A H7100 transmission electron microscopy
(TEM) (Hitachi High-Technologies Corp., Tokyo, Japan)
operating at 75 keV was used to collect TEM images of
the FSN-AuNPs. The average diameter of the AuNPs was
calculated using ImageJ software (http://rsb.info.nih.gov/ij/).
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The fluorescence spectra of OPA/2-ME-derivatized analytes were
obtained using a Hitachi F-7000 fluorometer (Hitachi, Tokyo,
Japan). The fluorescence images were recorded using a Coolpix
5400 digital color camera (Nikon, Tokyo, Japan).
Nanoparticle synthesis
We prepared citrate-capped AuNPs by means of the chemical
reduction of a metal salt precursor (hydrogen tetrachloroaurate,
HAuCl4) in the liquid phase.23 To achieve this, we rapidly added
38.8 mM of trisodium citrate (40 mL) to a solution of 1-mM
HAuCl4 (400 mL) that was heated under reflux. This heating
continued for an additional 15 min, during which time the color
of the solution changed to a deep red. TEM images (not shown)
confirmed that the diameter of the AuNPs is 13.1 nm, with
a standard deviation of 1.1 nm. The concentration of spherical
AuNPs was estimated by using the following equation:24
n ¼ 3m/4pr3s
where n is the number of AuNPs, m is the molar mass of Au in
the substance, r is the radius of the AuNPs, and s is the specific
gravity of AuNPs (19.3 g cm3). The values of m and r were
obtained by conducting inductively coupled plasma mass
spectroscopy (Perkin Elmer-SCIEX, Thornhill, ON, Canada)
and TEM measurements, respectively. Based on this result, we
estimated the concentration of the particles in the solution to
be 2.57 nM (1.55  1012 particles/mL). A solution of the
FSN-AuNPs was prepared upon the addition of 240 mL of 10%
FSN to 60 mL of 2.57 nM citrate-capped AuNPs. The resulting
mixture was stored at 4  C until further use. Although FSN-
AuNPs can not be reused after thiol extraction, the cost for
60 mL of 2.57 nM FSN-AuNPs is only ca. US$ 0.61. In this
study, 2.57 nM FSN-AuNPs denotes 1.0.
Sample preparation and detection
We prepared all standard solutions (1.0 mM) in deionized water
and then added 500 mL of 10.0 mM aminothiols to 500 mL of
FSN-AuNPs (2.0–24.0) or citrate-capped AuNPs (2.0–24.0),
which has been prepared in 80 mM phosphate solution at pH
range of 13.0. The mixtures were equilibrated at ambient
temperature for 30 min and then centrifugated at 17000 rpm for
10 min. Subsequently, the supernatant was carefully removed up
to a residual volume of 10 mL. The precipitates were resuspended
in a fresh prepared solution of 2-ME (5.0–200.0 mM; 100 mL) in
order to liberate the extracted aminothiols from the Au surface.
After 1 h, the released aminothiols were separated from the
precipitates by centrifugation at 17000 rpm for 10 min. The
resulting supernatant was placed in another 1.5-mL tube and
then derivatized with a solution containing OPA and NaOH. The
final concentrations of OPA and NaOH were 10.0–1000.0 mM
and 0.1–1.4 M. After 0–40 min, the fluorescence spectra of
OPA-derivatized aminothiols were measured using a fluorometer
when excited at 370 nm
In the quantitative analysis, we added 500 mL of aminothiols
(1.2–40.0 mM) to 500 mL of 40.0 FSN-AuNPs, which has been
prepared in 80 mM phosphate at pH 13.0. After 30 min, the
resulting solutions were centrifugated at 17000 rpm for 10 min
and then removed up to a residual volume of 10 mL.
Analyst, 2010, 135, 104–110 | 105

Subsequently, the precipitates were resuspended in 100 mL of
100.0 mM 2-ME. After 1 h, the supernatant were obtained by
centrifugation at 17000 rpm for 10 min and then derivatized with
a solution containing OPA and NaOH for 2 min. The final
concentrations of OPA and NaOH were 1.0 mM and 0.6 M. The
fluorescence spectra of OPA-derivatized aminothiols were
recorded using a fluorometer when excited at 370 nm.
Detection of total amount of HCys in urine
Urine samples were collected from a healthy female. To deter-
minate the total concentration of HCys in a urine sample, we
added 10 mL of 100.0 mM TCEP to 100 mL urine (Note that
TCEP can efficiently reduce HCys disulfide to generate HCys).
The mixtures were equilibrated at ambient temperature for
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10 min. We then prepared a series of samples by ‘‘spiking’’ them
with standard solutions (100 mL) of HCys in the range of 1.0 to
10.0 mM. Subsequently, we added these spiked samples (500 mL)
to a solution (500 mL) containing 40.0 FSN-AuNPs and 80 mM
phosphate (pH 13.0). The following steps were the same as those
described above in the quantification of standard solution of
HCys. On the other hand, the concentration of creatinine in urine
was measured by a TBA-200 FR automatic analyzer (Toshiba,
Tokyo, Japan).
Capillary electrophoresis
A commercial UV–vis absorbance detector (ECOM, Prague,
Czech Republic) was used to detect aminothiols while the
detection wavelength was set at 200 nm. Electrophoresis was
driven by a high-voltage power supply (Bertan, Hicksville, NY).
The high-voltage end of the separation system was put in
a laboratory-made plexiglass box for safety. Data acquisition
(10 Hz) and control were conducted by the use of DataApex
Software (DataApex, Prague, Czech Republic). The fused-silica
Scheme 1 Selective detection of HCys by the combination of FSN-AuNPs extraction and OPA/2-ME derivatization.
106 | Analyst, 2010, 135, 104–110
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capillaries (Polymicro Technologies, Phoenix, AZ) were 60-cm
long and had an i.d. of 75-mm (effective length: 40 cm). Prior to
analysis, capillaries were treated with 1.2% v/v PDDAC
overnight, resulting in reversed electroosmotic flow. Before
conducting separations, the capillary was filled with 1.2% v/v
PDDAC by syringe pumping (KD scientific, New Hope, PA) at
a flow rate of 1 mL min1. Note that PDDAC has been prepared
in 5.0 mM phosphate buffer at pH 1.0. Subsequently, the
released aminothiols were injected by hydrodynamic injection
at 20-cm height for 60 s. All separations were performed at
170 V cm1.
Results and discussion
Extraction of HCys by FSN-AuNPs
Scheme 1 shows that the AuNP-based approach for sensing
HCys can be divided into four major steps. First, the
FSN-AuNPs selectively capture HCys and Cys. Second, after
centrifugation and washing, HCys and Cys attached to the NP
surface are isolated from a mixture of analytes. Third, additional
2-ME removes HCys and Cys from the surface of the AuNPs
through ligand exchange between 2-ME and aminothiols.20
Finally, a solution of OPA and 2-ME selectively detects HCys in
the presence of Cys when excited at 370 nm. Note that the
fluorescence excitation and emission spectra of OPA/2ME-
derivatized HCys are entirely different from that of OPA/2-ME-
derivatized Cys (Fig. S1, ESI†). Table 1 shows that the reaction
of Cys with OPA/2-ME exhibits extremely weak fluorescence22
compared to HCys, GSH, and His. Additionally, a previous
study reports that the reaction rate between Cys and OPA/2-ME
is extremely slow.21 These results and previous studies suggest
that Cys does not interfere with the determination of HCys when
derivatized with OPA/2-ME (excited at 370 nm). To test whether
or not this approach is capable of sensing HCys, an experiment in
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Table 1 Relative fluorescence intensities (RFI%) of HCys and related

compounds derivatized with 0.1 mM OPA in the presence of 0.1 M 2-ME

RFIa Standard deviation (%)

HCys 100.0 3.5

Cys 0.4 2.1
GSH 63.8 2.6
Glu-Cys 177.8 4.6
Cys-Gly 0.1 1.9
His 89.3 3.4
a
Excitation at 370 nm.

this study utilized 1.0 FSN-AuNPs to selectively extract

10.0 mM HCys from a 40 mM phosphate solution (pH 13.0), and
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then used 0.1 M 2-ME to liberate the covalently attached HCys

from the Au surfaces. The released HCys was derivatized with
a solution of 0.1 M NaOH and 0.1 mM OPA. The fluorescence
spectrum of standard HCys derivatized with OPA/2-ME exhibits
an emission maximum at 485 nm when excited at 370 nm
(spectrum a in Fig. 1A).25 The released HCys derivatized with
OPA/2-ME reveal a similar fluorescence feature (spectrum b in
Fig. 1A), demonstrating that HCys detection can be conducted
by combining FSN-AuNPs extraction and OPA/2-ME derivati-
zation. Nevertheless, the fluorescence intensity of released HCys
is lower than that of standard HCys. This is probably because the
concentration of FSN-AuNPs is insufficient for the effective
extraction of HCys. To support this hypothesis, the supernatant
isolated from the centrifugation of a solution of FSN-AuNPs
and HCys was also reacted with OPA/2-ME. The strong
fluorescence intensity of the OPA derivative of the supernatant
reveals that a large number of HCys molecules remained in the Fig. 1 Comparison of fluorescence spectra of OPA/2-ME-derivatized
supernatant (Spectrum c in Fig. 1A). Note that 1.0 FSN- (A) HCys and (B) Cys. The supernatant and precipitate were obtained by
AuNPs can only extract 24.5% of the HCys from an aqueous the centrifugation of a solution containing 1.0 FSN-AuNPs and
solution. Because the concentration of 2-ME (0.1 M) is tremen- 10.0 mM aminothiols. To liberate aminothiols adsorbed on the NP
surface, the precipitate was resuspended in a solution of 0.1 M 2-ME. The
dously greater than that of HCys (10.0 mM), slow ligand
released aminothiols were isolated from the precipitate by the centrifu-
exchange between HCys and 2-ME on the surface of the AuNPs
gation. (a) standard aminothiols, (b) supernatant, and (c) released ami-
is impossible. nothiols were derivatized with a solution containing 0.1 mM OPA and
Next, under identical extraction conditions, another experi- 0.1 M NaOH for 10 min. FSN-AuNPs were prepared in 40 mM phos-
ment examined the fluorescence spectra of standard Cys, released phate solution at pH 13.0. The excitation wavelength was set at 370 nm.
Cys, and the supernatant (from the centrifugation of a solution
of FSN-AuNPs and Cys) when derivatized with OPA/2-ME. The
Effect of the concentration of FSN-AuNPs
OPA/2-ME-derivatized standard Cys had an extremely low
fluorescence intensity (Spectrum a in Fig. 1B), which may be the The results above imply that a higher concentration of
result of OPA reagent. Similar fluorescence spectra appear in the FSN-AuNPs is required to extract 1.0 mL of 10.0 mM HCys.
case sis of released Cys and the supernatant (Spectrum b and c in Thus, this study investigates the effect of the concentration of
Fig. 1B). These results show that OPA/2-ME can be used to FSN-AuNPs on the extraction efficiency of HCys. Fig. 2 shows
discriminate HCys from Cys when excited at 370 nm. A further that the fluorescence intensity (485 nm) of released HCys deriv-
experiment applied CE to examine whether or not FSN-AuNPs atized with OPA/2-ME gradually increases as the concentration
can be used to extract HCys and Cys. After extraction and 2-ME of FSN-AuNPs increases and reaches a plateau at 7.0 AuNPs.
liberation, 2-ME released-aminothiols were directly separated by This implies that 7.0 FSN-AuNPs are capable of completely
CE with UV absorbance. The electrophoregram a in Fig. S2 extracting 1.0 mL of 10.0 mM HCys. Accordingly, a single FSN-
(ESI†) shows three peaks corresponding to 2-ME, FSN, and AuNP (13-nm diameter) can adsorb approximately 560 HCys
HCys in the presence of 1.2% PDDAC. With the exception of the molecules. This study also examines whether or not the
Cys migration time, a similar electrophoregram was obtained for remaining aminothiols (Cys, GSH, Glu-Cys, and Cys-Gly) have
released Cys (electrophoregram b in Fig. S2, ESI†). Obviously, the same effect under identical extraction and derivatization
these results not only demonstrate the ability of FSN-AuNPs conditions. If the extraction of GSH and Glu-Cys by
to extract HCys and Cys, but also support the selectivity of FSN-AuNPs is successful, strong fluorescence signal should
OPA/2-ME reagent to HCys. appear through OPA/2-ME derivatization. However, when the

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Fig. 2 Effect of the concentration of FSN-AuNPs on the fluorescence

intensity at 485 nm of released aminothiols derivatized with OPA/2-ME.
The precipitate was collected by the centrifugation of a solution con-
taining 10.0 mM aminothiols and 1.0–12.0 FSN-AuNPs. The extraction
and derivatization conditions are the same as those in Fig. 1. FSN-
AuNPs were prepared in 40 mM phosphate solution at pH 13.0. The
excitation wavelength was set at 370 nm.
concentration of FSN-AuNPs increases from 1.0 to 12.0, the
fluorescence intensity for all tested aminothiols remains low
(Fig. 2). This implies that FSN-AuNPs have extremely low
affinity with GSH and Glu-Cys due to steric effects. Previous
studies report the same results.18,19 Moreover, because the
derivatization efficiency of Cys-Gly and Cys with OPA/2-ME is
extremely poor, the interferences of Cys-Gly and Cys for this
approach are negligible. According to these results, the concen-
tration of FSN-AuNPs for the complete extraction of 1.0 mL of
10.0 mM HCys is larger than 7.0. To show the advantage of
FSN-AuNPs over citrate-capped AuNPs, the extraction of each
aminothiol was conducted using different concentrations of
citrate-capped AuNPs. Fig. S3 (ESI†) shows that the fluores-
cence intensity (485 nm) of released HCy derivatized with OPA/
2-ME randomly changes as the concentration of citrate-capped
AuNPs increases. Similar results were observed in the case of
GSH and Glu-Cys. This may be attributed to that the fact that
high concentration of citrate-capped AuNPs was easily aggre-
gated in a high ionic strength solution. More importantly, this
result also indicates that citrate-capped AuNPs were not selective
to HCys.
Fig. 3 Fluorescence intensity at 485 nm of 2-ME released analytes
derivatized with OPA/2-ME. The precipitate was collected by the
centrifugation of a solution containing 1.0–100.0 mM analytes and 10.0
FSN-AuNPs. To liberate analytes adsorbed on the NP surface, the
precipitate was resuspended in a solution of 0.1 M 2-ME. The released
analytes were isolated from the precipitate by the centrifugation and then
derivatized with a solution containing 1.0 mM OPA and 0.6 M NaOH for
2 min. FSN-AuNPs were prepared in 40 mM phosphate solution at pH
13.0. The excitation wavelength was set at 370 nm.
complete extraction of HCys. Fig. 3 indicates that the combi-
nation of FSN-AuNPs extraction and OPA/2-ME derivatization
provides high selectivity for HCys over other aminothiols and
histidine; Fig. 3 also suggests that the selectivity of this approach
is more than 100-fold for HCys over other analytes.
The proposed method quantifies HCys by monitoring the
fluorescence intensity (485 nm) of OPA-derivatized HCys after
extracting various concentrations of HCys with 20.0
FSN-AuNPs. Note that the FSN-AuNPs concentration
increases to 20.0 to expand the dynamic range of the measur-
able concentration. As Fig. 4 indicates, the fluorescence intensity
at 485 nm increases as the concentration of released HCys

Selectivity, sensitivity, and application

This study examines the derivatization conditions for released

HCys in terms of fluorescence intensity and reaction time. Fig. S4
(ESI†) shows that the optimal concentrations of NaOH and
OPA for the derivatization of released HCys are 0.6 M and
1.0 mM, respectively. Under optimum extraction and derivati-
zation conditions, we evaluated the selectivity of our new
approach for HCys over aminothiols (Cys, GSH, Cys-Gly, and
Fig. 4 The quantification of HCys by the combination of 20.0 FSN-
Glu-Cys) and histidine. Since the precipitate (i.e., analyte- AuNPs extraction and OPA/2-ME derivatization. Inset: A plot of fluo-
attached AuNPs) is washed twice with distilled water, the pH of rescence intensity at 485 nm as a function of the HCys concentration. The
the solution in the extraction procedure does not interfere with concentration of FSN-AuNPs was 20.0. The error bars represent
the derivatization of analyte with OPA/2-ME. Moreover, the standard deviations based on five independent measurements. The other
FSN-AuNPs concentration was increased to 10.0 to ensure the extraction and derivatization conditions are the same as those in Fig. 3.

108 | Analyst, 2010, 135, 104–110 This journal is ª The Royal Society of Chemistry 2010

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Fig. 5 The quantification of total amount of HCys in urine by the
combination of 0.0 FSN-AuNPs extraction and OPA/2-ME derivati-
zation. Urine samples were spiked with standard solutions containing 0,
1.0, 2.0, 4.0, 6.0, 8.0, 10.0 HCys. Inset: A plot of fluorescence intensity at
485 nm as a function of the HCys concentration. The precipitate was
obtained by centrifugation of a solution containing spiked-urine samples
and 20.0 FSN-AuNPs. The error bars represent standard deviations
based on five independent measurements. The other extraction and
derivatization conditions are the same as those in Fig. 3.
increases. The inset in Fig. 4 shows a linear correlation between
the fluorescence intensity (485 nm) and the concentration of
HCys range from 0.6–40.0 mM. The limit of detection (LOD) is
180 nM at a signal-to-noise ratio of 3 for HCys, which is below
the maximum permissible limit of HCys in a healthy individual.4
Compared to the use of 40-nm FSN-AuNPs for the detection of
HCys19 the proposed method provides higher sensitivity and
larger linear range for the determination of HCys.
This study further applies this highly sensitive and selective
approach to the practical analyses of total amount of HCys in
urine samples. Although urine samples contain many other ami-
nothiols (Cys, GSH, and Cys-Gly), the interferences of GSH and
Cys-Gly are insignificant after pretreating urine samples with
FSN-AuNPs.26 Moreover, the quantum yield of Cys derivatized
with OPA/2-ME is extremely low and the excitation maximum of
Cys is totally different from that of HCys. Thus, the sensing of
total HCys using this approach should be straightforward. To
reduce HCys disulfide to HCys, the urine samples were pretreated
with a reducing agent, TCEP. Fig. 5 shows an apparent increase in
the fluorescence intensity at 485 nm after urine samples were
spiked with standard solutions containing different concentra-
tions of HCys. Plotting the fluorescence intensity at 485 nm
against the concentration of spiked HCys produces a linear (R2 ¼
0.9999) calibration curve. Standard addition shows that the
concentration of total amount of HCys in urine is 0.81 
0.12 mmol mol1 creatinine (n ¼ 3). Note that the concentration of
creatinine in the urine samples was 12.6 mM. The concentration
ranges obtained in this study are in good agreement with those
reported in the literature,3,26 suggesting that this approach is
suitable for routine urine assays in clinical studies.
Conclusion
This study reports a new assay for the selective and sensitive
detection of HCy using FSN-AuNPs for selective extraction of
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HCys and Cys and OPA/2-ME for selective derivatization
of HCys, respectively. Compared to previous studies on sensing
HCys,11–15,18,19 the proposed method has the distinct advantages
of high sensitivity (LOD ¼ 180 nM), greater linear range
(0.6–40.0 mM), greater selectivity (over 100-fold), and simplicity
(easy preparation of FSN-AuNPs). In the opinion of the authors,
the sensitivity of this method should be further improved by using
FSN-AuNPs to extract the large sample volume. Moreover, we
believe that the present approach holds great potential for the
analysis of HCys thiolactone25 and protein-bound HCys.4a
Acknowledgements
We would like to thank National Science Council (NSC 96-2113-
M-110-008-) and National Sun Yat-sen University-Kaohsiung
Medical University Joint Research Center for the financial
support of this study. We also thank National Sun Yat-sen
University and Center for Nanoscience & Nanotechnology for
the measurement of fluorescence spectrum.
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