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Journal of Chromatography A, 1274 (2013) 145– 150

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

jou rn al h om epage: www.elsev ier .com/ locat e/chroma

igh-performance liquid chromatography assay of cysteine and homocysteinesing fluorosurfactant-functionalized gold nanoparticles as postcolumnesonance light scattering reagents

unyan Xiaoa, Huiling Gaoa, Qipeng Yuana, Chao Lua,∗, Jin-Ming Linb

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, ChinaDepartment of Chemistry, Tsinghua University, Beijing 100084, China

r t i c l e i n f o

rticle history:eceived 11 October 2012eceived in revised form 6 December 2012ccepted 8 December 2012vailable online 17 December 2012

a b s t r a c t

Herein, a new postcolumn resonance light scattering (RLS) detection approach coupled with high-performance liquid chromatography (HPLC) was developed to detect cysteine and homocysteine. In theestablished system, the fluorosurfactant-capped gold nanoparticles (AuNPs) were first employed as post-column RLS reagents. The detection principle was based on the enhancement of RLS intensity of AuNPsupon the addition of cysteine/homocysteine. The RLS signals were detected by a common fluorescencedetector at �EX = �EM = 560 nm. The linear ranges for both cysteine and homocysteine were in the range of

eywords:ostcolumn reagentsold nanoparticlesesonance light scatteringigh-performance liquid chromatographyysteine

5.0–50 �M. The detection limits were 5.9 pmol for cysteine and 12 pmol for homocysteine at a signal-to-noise ratio of 3. HPLC separation and RLS detection conditions were optimized in detail. The applicabilityof the proposed method has been validated by detecting cysteine and homocysteine in human urinesamples. Recoveries from spiked urine samples were 95.0–103.0%.

© 2012 Elsevier B.V. All rights reserved.

omocysteine

. Introduction

Cysteine and homocysteine are vital biological thiols, whichidely occur in biological cells and tissues. They play an

rreplaceable role in numerous physiological processes, such asetoxification, metabolism, intracellular signal transduction andaintaining the balance between reduced and oxidized thiols

1–3]. Furthermore, levels of cysteine and homocysteine in organ-sm are related to several human diseases [4]. The deficiencyf cysteine is associated to liver damage and skin lesions [5,6].he altered level of homocysteine is implicated in cardiovascu-ar diseases and Alzheimer’s disease [7]. In addition, cysteinend homocysteine are very similar in structure and propertiesFig. 1). Abundant attentions have been paid to explore accuratend interference-free determination of cysteine and homocysteinen biological fluids, which is of great importance in physiology andlinical applications [8,9].

Gold nanoparticles (AuNPs) can be synthesized in a straightfor-

ard manner, possess tunable optical properties, exhibit excellent

iocompatibility after appropriate modification, and offer target-pecific platforms [10–12]. Therefore, AuNPs are one of the most

∗ Corresponding author. Tel.: +86 10 64411957; fax: +86 10 64411957.E-mail addresses: luchao@mail.buct.edu.cn, luchao20022002@yahoo.com.cn

C. Lu).

021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2012.12.016

versatile and widely researched materials for optical sensingincluding colorimetric, fluorescent, resonance light scattering,chemiluminescent and surface-enhanced Raman scattering tech-niques [13]. In general, the most popular and reliable method forproducing AuNPs is an aqueous synthesis by chemical reduction ofHAuCl4 with citrate [14]. Citrate-capped AuNPs are non-selectivitytowards specific targets, and instable in the presence of high ionicstrength and over a wide pH range. It needs to use some ligands(e.g., surfactants and single-stranded DNA) to improve stability andselectivity of AuNPs prior to the specific sensing step [15,16]. How-ever, it is a great challenge to realize such object on account ofthe difficulty of obtaining single target ligands. For the sake ofAuNP-based highly selective sensing, separation techniques suchas high-performance liquid chromatography (HPLC) and capillaryelectrophoresis (CE) may be alternative strategies.

In 2007, Zu’s group pioneered the use of fluorosurfactant-capped AuNPs as specific postcolumn colorimetric reagents forHPLC assay of cysteine and homocysteine in human urine andplasma samples [17]. Subsequently, the different ligand-cappedAuNPs have been successfully employed as HPLC/CE postcolumncolorimetric [18], chemiluminescent [19–21], electrochemilumi-nescent [22], and electrochemical [23] reagents for the detection

of a variety of analytes. These AuNP-based postcolumn detectionmethods have been successfully used for quantitation of analytes.However, each of these methods has advantages and disadvan-tages. Postcolumn colorimetric method offers simple operation,

146 Q. Xiao et al. / J. Chromatogr. A

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Fig. 1. Structures of cysteine and homocysteine.

ut it suffers from low sensitivity and narrow linear dynamicange [17,18]. Although postcolumn chemiluminescent methodxhibits high sensitivity, low noise and wide linear dynamic range,n additional chemiluminescence system (e.g., luminol-H2O2)ccompanied with complex procedures is required to generateensing signals [19–21]. As for postcolumn electrochemilumines-ent method, it is a challenge for researchers to design a properlectrochemiluminescent cell, which should not only assure theigh sensitivity of electrochemiluminescence, but also efficientlyliminate the high voltage electric field influence on electrochemi-uminescence [22]. Postcolumn electrochemical method may beisturbed by many factors, such as impurities and dissolved oxygen

n mobile phase, mobile phase temperature, and electrode contam-nation [23]. Therefore, there is currently an increasing interest inxploring novel AuNP-based postcolumn detection method.

Since the resonance light scattering (RLS) phenomenon wasrst reported by Pasternack in 1993 [24], RLS technique has beenroposed for the determination of DNA, proteins, metal ions, andrugs [25,26]. Recently, great attention has been focused on these of AuNPs as RLS probes by virtue of their extremely strong

ight scattering at the plasmon-resonance wavelength due to theollective oscillation of their conduction electrons [27–29]. RLSechnique based on self-assembly of AuNPs can improve sensitivity,nd thus it is becoming increasingly attractive in biochemical assay,harmaceutical analysis and biological imaging [25]. However,wing to its inherently low selectivity, development of specificLS assays on the basis of target-involved assembly of AuNPs

s still challenging and highly desirable. Fortunately, RLS tech-ique has been successfully incorporated with HPLC to achieveimultaneous determination of target analytes by forming ion-ssociation complexes between the separated components andolecular recognition probe [30–34]. Nevertheless, no case on the

se of AuNPs as HPLC postcolumn RLS molecular recognition probeas reported to date. A major obstruction leading to this situa-

ion was due to the fact that the aggregation/assembly of AuNPsenerally takes several to tens of minutes, making it difficult to beoupled with HPLC [17].

We recently utilized the fluorosurfactant-capped AuNPs as RLS probe to detect cysteine and homocysteine in biologicaluids by tuning the pH values of the gold suspensions [35].nder optimal conditions, the aggregation of AuNPs induced byysteine/homocysteine can be completed in 30 s; however, theynamic range of this RLS probe was narrow. In this work, wesed fluorosurfactant-capped AuNPs as postcolumn RLS reagentsor the assay of cysteine and homocysteine. Various experimen-al parameters for HPLC separation and postcolumn RLS detectionere optimized in order to achieve a highly sensitive and accurate

ssay of cysteine and homocysteine. Furthermore, the analyticalerits of the proposed method were demonstrated by detecting

ysteine and homocysteine in human urine samples with satisfac-ory results. To the best of our knowledge, this is the first report on

he use of nanoparticles as postcolumn RLS reagents. Our successrovides evidence that AuNPs as postcolumn RLS reagents may ben effective method to detect aminothiols and other target analytesy tuning ligands of nanoparticles.

1274 (2013) 145– 150

2. Experimental

2.1. Reagents and materials

All reagents were of analytical grade and used without fur-ther purification. All solutions were prepared with deionized water(Milli Q, Millipore, Barnstead, CA, USA). Hydrogen tetrachloroau-rate(III) trihydrate (HAuCl4·3H2O, 99.99%) and trisodium citrate(99%) were purchased from Acros (Geel, Belgium). Zonyl FSN-100(F(CF2CF2)1-7CH2CH2O(CH2CH2O)0-15H), cysteine (≥99%), homo-cysteine (>90%), tris(2-carboxyethl) phosphine (TCEP, ≥98%) andethylenediaminetetraacetic acid (EDTA, >99%) were purchasedfrom Sigma–Aldrich (St. Louis, USA). Methanol, acetonitrile, per-chloric acid (HClO4, 70–72%) and trifluoroacetic acid (TFA, ≥99.5%)were purchased from Beijing Chemical Reagent Company (Beijing,China). The pH of phosphate buffer solution (PBS) was adjusted withNaOH or HCl. 10 mM stock solutions of cysteine and homocysteinestandard substances were freshly prepared by dissolving their com-mercial crystal in deionized water and stocked at 4 ◦C until furtheruse, and their working solutions were freshly diluted their stocksolutions with deionized water. The HPLC mobile phase contain-ing 0.05% TFA was fresh daily prepared, filtered through 0.22 �mmembranes, and then degassed prior to use.

2.2. Apparatus

UV–visible spectra of spherical AuNPs were recorded using aUSB4000 miniature fiber optic spectrometer in absorbance modewith a DH-2000 deuterium and tungsten halogen light source(Ocean Optics, Dunedin, FL). The sizes and distribution of AuNPswere further confirmed through transmission electron microscope(TEM) measurements using a HITACHI-800 TEM from Hitachi(Tokyo, Japan). The RLS spectra and intensities were recorded bya Hitachi F-7000 fluorescence spectrophotometer equipped with a180 �L flow cell (Hitachi, Japan). The HPLC-RLS detection systemincluded a HPLC separation system and a postcolumn RLS detec-tion system. The HPLC separation system consisted of a ShimadzuLC-10AT pump (Shimadzu, Japan), a Genuine Rheodyne 7725i man-ual sample valve injector equipped with a 50 �L loop, a SunFireC18 guard column (4.6 × 20 mm, 5.0 �m particle size) and a GraceC18 analytical column (4.6 × 150 mm, 5.0 �m particle size) at roomtemperature. The postcolumn RLS detection system included a Shi-madzu LC-10AT pump (Shimadzu, Japan) to deliver the postcolumnreagents (fluorosurfactant-capped AuNPs), one mixing tee, a reac-tion system containing a reaction coil (0.5 mm i.d., 157 �L) and awater bath operated at 70 ◦C, and a Hitachi F-7000 fluorescencespectrophotometer equipped with a flow cell (180 �L).

2.3. Synthesis of AuNPs

All glassware for preparation of AuNPs was thoroughly washedwith freshly prepared aqua regia (HNO3:HCl = 1:3), rinsed exten-sively with deionized water, and then dried in an oven at 100 ◦C for2–3 h. 14 nm AuNPs were prepared according to the literature [17].Briefly, a 50 mL solution of 0.04% trisodium citrate was brought toa vigorous boil with stirring in a round-bottom flask fitted witha reflux condenser, and then 85 �L of 5% HAuCl4 was added tothe above solution. The solution was maintained at the boilingpoint with continuous stirring for 15 min and then the solution wascooled to room temperature with continued stirring. Then 400 �Lof 5% FSN was added. The suspension was stored at 4 ◦C until furtheruse. Assuming the spherical particles with a density equivalent to

that of bulk gold (19.30 g/cm3), the concentration of 14 nm AuNPswas calculated to be ∼2.9 nM. The TEM specimens were prepared bydepositing an appropriate amount of the fluorosurfactant-cappedAuNPs onto the carbon-coated copper grids, and excess solution

Q. Xiao et al. / J. Chromatogr. A 1274 (2013) 145– 150 147

Fig. 2. Schematic diagram of HPLC-RLS system for the determination of cysteine and homocysteine. The standard cysteine/homocysteine or sample solutions were separatedby isocratic elution; the mobile phase was 0.05% TFA, the concentration of fluorosurfactant-capped AuNPs was 2.9 nM, and the flow rates of TFA and fluorosurfactant-cappedA tively7 ine/hot

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The spectra were measured by scanning synchronously excitationand emission wavelengths from 500 to 750 nm on the Hitachi F-7000 fluorescence spectrophotometer. The results showed that RLSintensity can be increased with increasing cysteine concentration,

uNPs containing 100 mM PBS (pH 6.0) were 0.7 mL/min and 0.5 mL/min, respec725i manual sample valve injector equipped with a 50 �L loop; S, standard cysteemperature of heater was set to 70 ◦C; PMT, photomultiplier tube; W, waste.

as wicked away by a filter paper. Then the grid was subsequentlyried in air.

.4. HPLC-RLS system

Fig. 2 illustrated the flow path of the postcolumn HPLC-RLSetection system. 0.05% TFA served as mobile phase and wasumped by the high pressure pump (P1) at 0.7 mL/min. Standardixture of cysteine and homocysteine or human urine samplesere injected into the mobile phase stream through the manual

ample valve injector and then separated by column. Then the sep-rated solution mixed with 2.9 nM fluorosurfactant-capped AuNPsontaining 100 mM PBS (pH 6.0) in the three-way pieces. Note thathe use of 100 mM PBS can keep the suitable ionic strength andH value of the gold colloidal solution for the interaction betweenuNPs and aminothiols. Fluorosurfactant-capped AuNPs contain-

ng 100 mM PBS (pH 6.0) was pumped by another high pressureump (P2) at 0.5 mL/min. The mixture was heated in a reactionoil, which was immersed in the 70 ◦C water bath. The intensitiesf RLS were obtained by synchronously setting the excitation andmission wavelength at 560 nm (�EX = �EM = 560 nm). The excita-ion and emission bands widths were set to 5.0 nm with scan speedf 1200 nm/min, and the PMT voltage was 500 V. The enhancementf the RLS intensities were calculated by �I = I − I0, where I0 washe RLS intensity in the absence of cysteine/homocysteine and I washe RLS intensity in the presence of cysteine/homocysteine.

.5. Sample collection and pretreatment

Human urine samples were collected from two healthy volun-eers, and the analysis was conducted immediately after the sampleollection. The standard addition was carried out by spiking a cer-ain amount of cysteine/homocysteine standard solution to urineamples. To 100 �L of urine sample in a centrifuge tube, 50 �L of.1 M EDTA and 5.0 �L of 0.2 M TCEP in pH 6.0 PBS were added.

he mixture was kept at 60 ◦C for 30 min. After cooling to roomemperature, the sample was gently vortex-mixed with 20 �L of.0 M HClO4 solution, put aside at room temperature for 10 min, andhen centrifuged at 13,000 rpm for 10 min. The clear supernate was

; the volume of flow cell was 180 �L; P1 and P2 were two high press pumps; V,mocysteine or human urine samples; G, guard column; A, analytical column; the

filtered through a 0.22 �m filter and diluted when required beforeinjecting 50 �L of the sample into the HPLC system.

3. Results and discussion

3.1. Detection wavelength and slit width

The RLS spectra of fluorosurfactant-capped AuNPs in the pres-ence of different concentrations of cysteine were shown in Fig. 3.

Fig. 3. Resonance light scattering spectra of fluorosurfactant-capped AuNPs con-taining 100 mM PBS (pH 6.0) in the presence of different concentrations of cysteine.The concentrations of cysteine were (1) 0 �M; (2) 1.0 �M; (3) 3.0 �M; (4) 5.0 �M;(5) 7.0 �M; (6) 12.0 �M. Inset, TEM images of fluorosurfactant-capped AuNPs in theabsence (A) or presence (B) of 12 �M cysteine.

148 Q. Xiao et al. / J. Chromatogr. A 1274 (2013) 145– 150

Fig. 4. Effects of the reaction conditions on the HPLC-RLS system in the presence and absence of cysteine/homocysteine: (A) concentration of fluorosurfactant-capped AuNPs:the flow rates of TFA and AuNPs containing 100 mM PBS (pH 6.0) were 0.7 mL/min and 0.5 mL/min, respectively; the reaction temperature was 70 ◦C; (B) the incubationtemperature: the concentration of fluorosurfactant-capped AuNPs was 2.9 nM; the flow rates of TFA and fluorosurfactant-capped AuNPs containing 100 mM PBS (pH 6.0)w n of flA e was6 of TF

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ere 0.7 mL/min and 0.5 mL/min, respectively; (C) flow rate of TFA: the concentratiouNPs containing 100 mM PBS (pH 6.0) was 0.5 mL/min; the incubation temperatur.0): the concentration of fluorosurfactant-capped AuNPs was 2.9 nM; the flow rate

nd reached the maximum at a wavelength of about 560 nm.herefore, 560 nm was chosen as the optimum excitation and emis-ion wavelength in following experiments. Note that the largeggregates could be formed in the presence of 12 �M cysteinesee inset in Fig. 3), which was attributed to the production oftrong RLS signals [36,37]. Furthermore, excitation and emissionlit widths were also important parameters. When the two slitidths were narrower than 5.0 nm, the RLS spectra would be dis-

orted and the RLS intensity was decreased. On the other hand, theroader slit widths (>10 nm) might contain stray light [38]. Accord-

ngly, 5.0 nm was selected as the best slit width for excitation andmission.

.2. Postcolumn reaction coil length

The effect of postcolumn reaction coil length was exam-ned in the range of 30–240 cm. The RLS intensity graduallyncreased with an increase in the length of reaction coil up to0 cm. Moreover, cysteine and homocysteine were completelyeparated at 80 cm reaction coil. However, the RLS intensity

ecreased if the postcolumn reaction coil used was longer than0 cm, along with the broadening and tailing of chromatographiceaks. Therefore, a reaction coil of 80 cm was chosen in furtherxperiments.

uorosurfactant-capped AuNPs was 2.9 nM; the flow rate of fluorosurfactant-capped 70 ◦C; (D) flow rate of fluorosurfactant-capped AuNPs containing 100 mM PBS (pHA solution was 0.7 mL/min; the incubation temperature was 70 ◦C.

3.3. AuNPs concentration

As the postcolumn RLS reagents for assay of cysteine and homo-cysteine, the concentration of fluorosurfactant-capped AuNPstremendously affected the intensity of RLS. In this study, the con-centration of fluorosurfactant-capped AuNPs was investigated inthe range of 1.5–2.9 nM. It can be seen from Fig. 4A, the intensity ofRLS of fluorosurfactant-capped AuNPs increased with an increase inthe concentration of fluorosurfactant-capped AuNPs. In view of theconsumption of the reagents and simplicity of the experimentalprocedures, the initial 2.9 nM concentration of fluorosurfactant-capped AuNPs was used throughout this study in view of the RLSintensity and the operation simplification (no need to dilute orconcentrate the as-prepared AuNPs).

3.4. Incubation temperature

Fast kinetics rate plays a key role in ensuring the overall suc-cess of HPLC postcolumn reaction. It has been reported that theaggregation rate of fluorosurfactant-capped AuNPs induced bycysteine/homocysteine was highly dependent on the incubation

temperature [35,39]. In this work, the influence of incubationtemperature in the range of 20–70 ◦C on the RLS intensity offluorosurfactant-capped AuNPs was examined (Fig. 4B). It wasshown that the aggregation rate was slow when the incubation

Q. Xiao et al. / J. Chromatogr. A 1274 (2013) 145– 150 149

Fig. 5. Typical HPLC chromatogram of a mixture of standard cysteine andhomocysteine (5.0 �M for each). The mobile phase was 0.05% TFA; the concen-tfli

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Fig. 6. (A) HPLC chromatogram of a human urine sample. The pretreated samplesolution was diluted three times with water prior to HPLC injection. (B) HPLC chro-

TA

ration of fluorosurfactant-capped AuNPs was 2.9 nM; the flow rates of TFA anduorosurfactant-capped AuNPs were 0.7 mL/min and 0.5 mL/min, respectively; the

ncubation temperature was 70 ◦C.

emperature was lower than 50 ◦C. However, when the incubationemperature was higher than 70 ◦C, a slight color change occurred,ndicating that fluorosurfactant-capped AuNPs became unstable.herefore, the postcolumn reaction of this system was incubatedt 70 ◦C.

.5. Flow rate selection

The flow rate of the mobile phase has a very important effectn the separation. When the flow rate of TFA was 0.7 mL/min, cys-eine and homocysteine could be separated completely, and theLS intensity was the strongest (Fig. 4C). However, when the flowate of TFA was larger than 0.7 mL/min, the sensitivity decreased.herefore, the optimal flow rate of TFA was selected as 0.7 mL/min.oreover, the flow rate of the postcolumn AuNPs was also opti-ized in the range of 0.1–0.9 mL/min (Fig. 4D). When the flow rate

f AuNPs was larger than 0.5 mL/min, the RLS intensity remainedonstant. Therefore, 0.5 mL/min was selected as the optimal flowate in this work.

.6. Assay validation

Under the optimum conditions, the colloidal solution ofuorosurfactant-capped AuNPs was employed as novel post-olumn RLS reagents for the determination of cysteine andomocysteine. Fig. 5 showed that cysteine and homocysteine wereell resolved at 2.6 and 3.8 min retention times, respectively. The

esults revealed that the RLS intensity increased linearly with theoncentration of cysteine/homocysteine in the range of 5.0–50 �M.he corresponding regression coefficient for cysteine and homocys-eine were 0.9977 and 0.9989, respectively. The detection limits

able 1nalytical results of human urine samples.

Samples Analytes Measured (�M)a

Urine 1 Cysteine 106.0 ± 3.7

Homocysteine 7.1 ± 0.6

Urine 2 Cysteine 112.3 ± 0.5

Homocysteine 4.0 ± 0.1

a Mean ± SD of three measurements.

matogram of a collected human urine sample spiked with the standard cysteine(3.0 �M) and homocysteine (30 �M). Experimental conditions were the same asthose described in Fig. 5.

were 5.9 pmol for cysteine and 12 pmol for homocysteine at asignal-to-noise ratio of 3. The precision and repeatability of theproposed method were evaluated by five replicate injections of themixed standard solution, and the relative standard deviation (RSD)was 2.6%.

3.7. Application to human urine samples

The proposed strategy was also applied to the detection of cys-teine and homocysteine in human urine samples. The urine samplesfrom two healthy people were pretreated as described above. Fig. 6illustrated the typical chromatogram of the human urine, along

with the sample spiked with a known amount of standard cysteineand homocysteine. It was obvious that cysteine and homocys-teine in the human urine were well-separated using the proposedHPLC-RLS system, and their retention times were close to those

Added (�M) Found (�M)a Recovery (%)a

50 51.5 ± 1.6 103.0 ± 3.2100 97.1 ± 1.9 97.1 ± 1.9

3 3.0 ± 0.1 100.0 ± 3.310 9.5 ± 0.4 95.0 ± 4.060 62.5 ± 0.2 104.2 ± 0.390 87.6 ± 0.1 97.3 ± 0.1

7 6.7 ± 0.2 95.7 ± 2.99 9.1 ± 0.1 101.1 ± 1.1

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f the standard cysteine and homocysteine, respectively. The con-entrations of cysteine and homocysteine in human urine samplesere obtained using a standard addition method and the resultsere summarized in Table 1, which were in accordance with those

btained by previous studies [35,40]. Also, the recoveries for cys-eine/homocysteine in spiked samples were found to be between5.0% and 103.0%. The obtained results indicated that the proposedethod was efficient and reliable.

. Conclusion

In summary, a novel HPLC-RLS assay of cysteine and homo-ysteine in human urine samples has been developed by usinguorosurfactant-capped AuNPs as postcolumn RLS reagents. To theest of our knowledge, this is the first report on using nanoparti-les as postcolumn RLS reagents to detect low molecular weightminothiols. Unfortunately, the sensitivity of the proposed methodas probably limited by using the large volume of the commer-

ial flow-through cell (180 �L). Attention is drawn to the improvedensitivity which can be obtained with a home-made substitute.

e anticipate that various functionalized nanoparticles as postcol-mn RLS reagents have much potential in a wide variety of sensingpplications.

cknowledgements

This work was supported by the National Natural Science Foun-ation of China (21077008 and 20975010), the Program for Newentury Excellent Talents in University (NCET-11-0561), the 973rogram (2011CBA00503) and the Fundamental Research Fundsor the Central Universities (ZZ1230).

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