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Ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 337–342
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction Ling-Ling Wang ⇑, Sheng Yu, Meng Yu College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" TiO2–Gr nanocomposite was used as
SPE adsorbent. " A novel spectrofluorimetry for
S-nitrosothiols determination was developed. " The developed method has low detection limit and wide linear range.
a r t i c l e
i n f o
Article history: Received 8 June 2012 Received in revised form 18 July 2012 Accepted 7 August 2012 Available online 23 August 2012 Keywords: S-nitrosothiols Spectrofluorimetric determination Solid-phase extraction TiO2–graphene nanocomposite
a b s t r a c t This present work describes the ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction based on a novel adsorbent TiO2–graphene nanocomposite. 1,3,5,7Tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence is used as fluorescent probe for S-nitrosothiols label. The procedure is based on the fluorescent probe selective reaction with S-nitrosothiols to form highly fluorescent product, its extraction to the TiO2–graphene-packed SPE cartridge and spectrofluorimetric determination. The experimental variables affecting the extraction procedure, such as the type of the eluent and its volume, sample pH, and sample volume, have been studied. Under the optimized extraction conditions, the method showed good linearity in the range of 0.5– 100 nM. The limit of detection was 0.08 nM (signal-to-noise ratio = 3). Relative standard deviation was 2.5%. The developed method was applied to the determination of S-nitrosothiols in human blood samples with recoveries of 92.0–104.0%. This work revealed the great potentials of TiO2–graphene as an excellent sorbent material in the analysis of biological samples. Ó 2012 Elsevier B.V. All rights reserved.
Introduction The obligatory role of nitric oxide (NO) in the regulation of cardiovascular system has been widely recognized. However, NO is known to be extremely unstable and susceptible to inactivation by heme iron, non-heme iron, superoxide anion, oxygen, and other biochemical species [1]. Indeed, it has been suggested that NO could be stabilized by covalent bonding with thiols such as glutathione, cysteine, albumin, and hemoglobin [2,3], forming S-nitrosocysteine (SNOCys), S-nitrosoglutathione (SNOGSH), S-nitrosoalbumin, and ⇑ Corresponding author. Tel.: +86 371 6390811. E-mail address: [email protected] (L.-L. Wang). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.08.045
S-nitrosohemoglobin, respectively. Therefore, accurate evaluation of S-nitrosothiols (SNOs) in circulating blood is of great interest in assessing the in vivo regulatory state of NO/cGMP system not only by the endothelial NO, but also by NO-releasing vasodilators in clinical use such as glyceryl trinitrate [4]. Some analytical methods for the determination of RSNO have been developed. These mainly include spectrophotometry [5], chemiluminescence [6], electrometric methods [7], chromatography [8] and spectrofluorimetry [9,10], etc. However, the extremely low concentrations in biological samples and the complexity of their matrices make determination of SNOs challenging tasks. Because of this, there is a critical demand for rapid and simple preparation techniques especially extraction
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techniques, for evaluating and monitoring SNOs from different biological samples at trace level. Solid-phase extraction (SPE) is an efficient sample pre-treatment method, routinely used for the extraction of compounds from liquid or solid matrices. SPE approach, in which adsorbents have been treated as media for the retaining of target compounds, followed by selective elution, has been broadly utilized due to the advantages of less organic solvents, no emulsion formation like in liquid–liquid extraction, and short time, etc. [11]. The choice of appropriate adsorbent is a critical factor to obtain good recovery and high enrichment factor in SPE procedure. Graphene (Gr) is a fascinating carbon nanomaterial, which posses a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice. It has a large specific surface area (theoretical value 2630 m2/g) which suggests a high sorption capacity [12]. Furthermore, special structure makes both sides of the planar sheets of Gr are available for molecule adsorption. Actually, Gr and its composite have been reported using as adsorbent in SPE for different compounds enrichment [13–17]. However, many of the interesting and unique properties of Gr can only be realized after it is integrated into more complex assemblies [18]. Moreover, two-dimensional plane structure of Gr provides a vast platform for loading various nanoparticles, offering a new way to develop catalytic, magnetic, and optoelectronic materials [19]. It has also been reported that the integration of carbon-based materials and metal oxide usually shows synergistic effects in applications [20], so there is a reason to expect the integration of Gr and other nanoparticles has the similar effect on the SPE of SNOs. TiO2 nanoparticles is reported having large surface area and containing much ionexchangeable OH groups in the interlayer and surface [21], and it has gained great interests in analytical chemistry because of its high chemical stability, durability, corrosion resistance, non toxicity and cost effectiveness. Micro-scaled TiO2 has been used as stationary phase in HPLC applications [22] as well as a solid phase extraction adsorbent for selective extraction of phosphopeptides [23]. TiO2 were demonstrated to be excellent adsorbents of inorganic cations and anions as well as organic compound [24]. Spectrofluorimetric method of determination has several advantages including low detection limit, sensitivity, selectivity, low cost and less time consuming. Difluoroboradiaza-s-indacenes (BODIPY) has advantages of high extinction coefficients, high fluorescence quantum efficiency and stability to light [9]. It has been reported that Hg2+ can effectively displace NO from GSNO, and then NO reacts with o-phenylenediamine to form fluorescent derivative [9]. 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABO) is an excellent fluorescent probe [25]. Its self-fluorescence is very weak, and the quantum efficiencies can increase more than 100 times after reaction with NO. In this work, a novel method was developed for the ultrasensitive and selective determination of SNOs in biological samples using fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABO) derivatization followed by SPE and spectrofluorimetric detection. TiO2–Gr was used as SPE adsorbents. Several key influence parameters were investigated in detail for good SPE efficiency. The method was demonstrated to be applicable for the analysis of SNOs in human blood samples. Experimental Apparatus All fluorescence measurements were performed on a Cary Eclipse Fluorescence Spectrophotometer (Varian, USA) with 1-cm
path length quartz cells. Instrument excitation and emission slits both were adjusted to 5 nm. The pH value of solution was measured using a pHS-3C meter (Shanghai Leici Equipment Factory, China). Scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 scanning electron microscope. The SPE cartridges (1 mL) were obtained from Agilent (Agilent, Palo Alto, CA, USA). Reagents 1,3,5,7-Tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)difluoroboradiaza-s-indacence (TMDCDABO) was synthesized according to the references [25,26]. The stock solution of 1 103 mol L1 TMDCDABO was prepared in methanol (MeOH). Glutathione (GSH) (reduced form, free acid 98–100%) was obtained from Shanghai Chemicals (Shanghai, China). Phosphate buffered saline (PBS) consisted of 10.0 g L1 NaCl, 0.25 g L1 KCl, 1.44 g L1 Na2HPO4, 0.25 g L1 KH2PO4 and adjusted to pH 7.0 [25]. Unless otherwise specified, all reagents were of analytical reagent grade used without further purification and all solutions were prepared from double-distilled water. Preparation of SNOs SNOs were prepared fresh for each experiment by incubating 100 mM thiol (GSH) with 100 mM sodium nitrite in the presence of 250 mM HCl and 0.1 mM EDTA for 30 min. The solutions rapidly turned red upon exposure to water, forming the corresponding SNOs. The stock solutions were stored on ice. The final concentrations were determined using the reported extinction coefficients at 334–338 nm for SNOGSH (e334nm = 780 mol1 cm1) [27]. Preparation of TiO2–Gr nanocomposite Graphene oxide (GO) was prepared from graphite powder by the modified Hummers method [28]. Graphite was added in a mixture containing 12 mL concentrated H2SO4, 2.5 g K2S2O8 and 2.5 g P2O5. The solution was heated to 80 °C and kept stirring for 5 h. The mixture was diluted with deionized water (500 mL). The product was obtained by filtering using 0.2 lm Nylon film and dried naturally. The product was re-oxidized by Hummers and Offeman method to produce the graphite oxide. Exfoliation was carried out by sonicating 0.1 mg mL1 graphite oxide dispersion for 1 h. TiO2– Gr nanocomposite was prepared according to the reference [29]. In short, 0.2 mL of Ti(OiPr)4 was added to the GO suspension (1 mg mL1) and ultrasonicated for 1 h. The mixture was then transferred to a 25-mL Teflon-sealed autoclave and kept at 130 °C for 12 h. The product was isolated by filtration and rinsed thoroughly with water and ethanol, respectively. The product was then dried in vacuum. The TiO2–Gr nanocomposite was obtained in the form of black powder. Derivatization procedures Hg2+ can effectively displace NO from GSNO, and then NO reacts with TMDCDABO to form TMDCDABO derivative. The derivatization procedures were according to the reference [10]. 2 mL 2.0 105 mol L1 TMDCDABO was transferred to a 10 mL volumetric flask. Then 2 mL PBS, 0.05 mL 5 104 mol L1 SNOGSH and 1 mL of 1 103 mol L1 Hg2+ solution were added. The mixture was diluted to the mark with water and kept at 30 °C for 15 min. The chemical structure of TMDCDABO and its reaction with SNOGSH are shown in Fig. 1.
L.-L. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 98 (2012) 337–342
NH2
NH2 O
O OH N S
NH H N
O
O
O
O Hg2+
O OH
OH HS
NH H N
O
+
NO
OH O
NH2
HN N N
NH2 H3C
339
CH3
CO2C2H5 N N B H3C F F CH3
NO
H3C
CH3
C2H5O2C
C2H5O2C
O2
CO2C2H5 N N B H3C F F CH3
Fig. 1. The reaction of TMDCDABO with SNOGSH.
Solid phase extraction procedure and spectrofluorimetric analysis TiO2–Gr (20 mg) was added in a 1 mL SPE cartridge using an upper frit and a lower frit to avoid adsorbent loss. Prior to extraction, the cartridge was preconditioned with 5 mL MeOH and 5 mL water successively. The sample solution (10 mL) was then passed through the cartridge. The analytes retained on the cartridge were subsequently eluted with 1 mL MeOH. The eluate was collected and the fluorescence was measured at kex/kem = 500/510 nm against a reagent blank carried through the same procedure except for the addition of SNOGSH.
recovery was defined as the percentage of total analyte which was extracted to SPE column.
Extraction recovery ¼ ½ðC 0 V 0 Þ ðC 1 V 1 Þ 100=ðC 0 V 0 Þ ð1Þ where C1 and C0 are the concentration of the analyte in the final and the initial concentration in the sample, respectively. V1 and V0 are the volumes of the samples involved.
Results and discussion Samples preparation
Morphology of graphene
Human blood samples were collected from free volunteers. All samples were obtained by venipuncture using sodium citrate as an anticoagulant and kept on ice in the dark. After bubbling oxygen for 10 min, the derivatization procedures were performed as described above. Subsequently, the mixture was centrifuged for 10 min at 3000 rpm and the supernatant was collected and determined by SPE-spectrofluorimetry method.
The morphology of the obtained Gr and TiO2–Gr was observed by SEM (Fig. 2). Fig. 2A shows the SEM image of Gr, revealing the typical crumpled and wrinkled Gr sheet structure. Fig. 2B presents the SEM image of TiO2–Gr composite. It shows the TiO2 nanoparticles are uniformly and compactly embedded on the Gr substrate.
Extraction recovery
In order to obtain the maximal extraction efficiency, several factors such as the elution solvent, the sample solution volume and pH value, flow rate of sample solution were optimized. All the optimization experiments were conducted three times.
One main parameter has been employed for the evaluation of the proposed configuration, namely: extraction recovery. Extraction
Optimization of the solid phase extraction procedure
Fig. 2. SEM image of Gr (A) and TiO2–Gr (B).
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120
80
Recovery (%)
Recovery (%)
100
60 40
A
100
80
60
20 40
0 Hexane
DCM
ACN
MeOH
0.5
1.0
Elution solvent
1.5
2.0
2.5
3.0
Elution volume (mL)
Fig. 3. Effect of elution solvent on recoveries. Eluent volume: 1 mL; sample flow rate: 2 mL min1; sample pH 7.0; sample volume: 10 mL.
110
B
Effect of flow rate of sample solution through the cartridge Flow rate of aqueous sample solution always has a significant impact in the SPE procedure, because the sample flow rate affects both the recoveries of analytes and loading time in a SPE system. The influence of sample flow rate was investigated over the range of 1–5 mL min1 with the other conditions kept constant. Fig. 4B shows that 2 mL min1 is optimal flow rate which is selected for further use in the following experiment. Effect of sample pH Sample pH plays an important role in the SPE procedure and affects the extraction efficiency. pH factor determine the surface charge of the TiO2–Gr nanoparticles. The –OH and –OOH groups can be protonated in acidic solution then TiO2–Gr has positive surface charge. However, in alkaline media the –OH and –OOH groups can be deprotonated and they have negative surface charge. The effect of sample pH on the recoveries of SNOGSH derivative was examined in a range of 4–9. The highest recovery is obtained at pH 7 (Fig. 4C). The analyte SNOGSH derivative is electric neutrality,
Recovery (%)
100 90 80 70 60 50 1
2
3
4
5 -1
Flow rate (mL min ) 110
C
100
Recovery (%)
Effect of elution solvent The type of elution solvent is important for the extraction efficiency [28]. The TiO2–Gr-packed cartridges were first washed thoroughly with water and various organic solvents including MeOH, acetonitrile (ACN), tetrahydrofuran (THF), and hexane, ethanol, acetone, dichloromethane. No visible adsorbent losses were observed. Four solvents with different polarities were tested: hexane, dichloromethane (DCM), acetonitrile (ACN) and MeOH. As shown in Fig. 3, MeOH yields the higher recovery (97.2%) than that of ACN (82.3%), because protic solvent (i.e., MeOH) can elute the polar derivative that may have hydrogen bonding with the hydroxyl groups on the TiO2–Gr surface more effectively than aprotic solvent (i.e., ACN). DCM also cannot satisfactorily elute the analyte with the recovery of 78.1%. Hexane has a poor eluting ability toward SNOGSH derivative (recovery 45.4%). Therefore, MeOH was selected for elution of analytes from the TiO2–Gr-packed SPE cartridges. The volume of the eluent has a great effect on the elution performance and efficiency. To find out the required MeOH volume to recover all the analytes from TiO2–Gr packed cartridge, eluent volumes in the range of 0.5–3 mL were tested. From Fig. 4A, it suggested that SNOGSH derivative needed a volume of eluent more than 1 mL to get the best recoveries. Thus, to achieve complete elution of the seven analytes, 1 mL MeOH was utilized in following experiments.
90 80 70 60 50 40 4
5
6
7
8
9
pH Fig. 4. Effects of elution volume (A), sample flow rate (B) and sample pH (C) on the recoveries. Other conditions are the same in Fig. 3.
and the most SNOGSH derivative is adsorbed on the SPE adsorbent at pH 7. So pH 7 was chosen as the pH of the sample solutions. Effect of sample volume To obtain reliable analytical results and high enrichment efficiency, it was necessary to obtain the breakthrough volumes in the SPE process. Different volumes (1, 3, 5, 10, 20 mL) of samples were investigated three times for the breakthrough volume. The results showed that the breakthrough volume of SNOGSH derivative was more than 20 mL at least. In practice, the sample volume was chosen according to the required sensitivity and the time acceptable for a whole analysis. Generally, further increasing of
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the sample volume was not desirable for routine analysis since the total time needed for one analysis would be longer. So a sample volume of 10 mL was selected for subsequent analysis with satisfactory sensitivity. Effect of amount of cartridge packing The efficiency of the TiO2–Gr amount on the recovery of analytes was studied. Four solid phase amounts were tested (5, 10, 20 and 30 mg) for the preconcentration of SNOGSH derivative. Obtained results showed insignificant differences in recoveries among the different amounts of cartridge packing. The highest recovery was obtained when 20 mg TiO2–Gr was used. Therefore, the TiO2–Gr amount of 20 mg was recommended for the extraction of SNOGSH derivative.
Table 1 Analytical characteristics of the developed method. Analytical parameters
Without SPE
With SPE
Linear range (nM) Regression equation Detection limit (nM) Correlation coefficient (R) Relative standard deviation (n = 6)
10–120 Y = 2.56 X + 20.439 1.0 0.9994 1.8
0.5–100 Y = 8.20 X + 45.21 0.08 0.9992 2.5
Excitation and emission slits both were 5 nm; kex/kem = 500/510 nm.
Table 2 Analytical results of the blood samples (n = 6). Samples
Added (nM)
Found (nM)
Recovery
RSD (%)
1
0 1 10 60 0 1 10 60 0 1 10 60
9.26 10.18 18.94 67.58 10.12 11.14 19.68 67.90 9.75 10.79 19.57 71.19
– 92.0 96.8 97.2 – 102.0 95.6 96.3 – 104.0 98.2 102.4
2.8 1.6 3.2 2.4 4.1 4.3 3.2 3.1 2.8 2.6 3.6 3.2
Comparison study The present results with TiO2–Gr, as new sorbent for the SPE, was compared to Gr and traditional method using C18 silica sorbent. SNOGSH derivative were extracted from by Gr and C18 procedure as the same as the TiO2–Gr. Fig. 5 shows that the extraction recovery of TiO2–Gr is the highest than that of Gr and C18. Although the recoveries obtained by TiO2–Gr was close to that by Gr, the elution of the analytes from the Gr-packed SPE cartridge consumed more time than from TiO2–Gr-packed SPE cartridge. The reason may be Gr is prone to congregate, and this hinders the objective molecules elution. Furthermore, it is necessary to take into account the cost of adsorbent. C18 silica sorbents are more expensive than the TiO2–Gr laborated with the simple hydrothermally method. Method validation To validate the applicability of the proposed SPE method using the TiO2–Gr sorbent, linearity and limit of detection (LOD) were assessed using the optimum extraction conditions. Table 1 shows that good linearity is obtained for SPE extractions using the TiO2–Gr sorbent with coefficient of determination (R = 0.9992). The LOD is 0.08 nM, which is calculated at a signal to noise ratio (S/N) of 3. The repeatability (RSD) is 2.5%. These values were better than that for the spectrofluorimetry without SPE procedure (Table 1). The TiO2–Gr cartridge was found practically reusable for all the extractions performed. The potential regeneration and stability of the TiO2–Gr cartridge were studied up to at least 30 adsorption-elution cycles. The column was washed with 5 mL of MeOH followed by 5 mL deionized water after each extraction. It was observed that TiO2–Gr SPE could
3
Elution solvent: MeOH; eluent volume: 1 mL; sample flow rate: 2 mL min1; sample pH 7.0; sample volume: 10 mL; excitation and emission slits both were 5 nm; kex/kem = 500/510 nm.
be repeatedly used (up to at least 30 adsorption-elution cycles) without significant loss of uptake capacity in the extraction of SNOGSH derivative. The interday precisions (RSDs) after 25 cycles of use were 3.2%. Furthermore, the cost of producing the TiO2–Gr was low and the process involve was simple and easily performed. Sample analysis The proposed method was applied to the determination of trace-level S-nitrosothiols in the blood samples collected from free volunteers. All samples were analyzed for six times using the same recommended procedure and conditions. The results are shown in Table 2. The recoveries ranged from 92.0% to 104.0% and the RSD from 1.6% to 4.3%, which confirmed the feasibility and reliability of the present method. Conclusions TiO2–Gr has been successfully applied as sorbent for SPE coupled with spectrofluorimetry for the determination of S-nitrosothiols from blood samples. LOD at about part per trillion level Snitrosothiols in blood samples was achieved using this nanocomposite. The SPE method presented here offers an interesting and effective option for the analysis of S-nitrosothiols and other spectrofluorimetry-amenable trace biomoleculars in similar sample types or similar analytes from other sample types. A comparison of TiO2–Gr SPE with commercial C18 SPE and Gr SPE indicates that TiO2–Gr sorbent is better in terms of recovery. TiO2–Gr sorbent is also cost effective and easy to prepare. The high surface area of the material facilitate in the adsorption process and its increase in sensitivity.
100
Recovery (%)
2
80 60 40 20 0 TiO2-Gr
Gr
C18
Fig. 5. Effect of adsorbent type on recoveries. Other conditions are the same in Fig. 3.
Acknowledgments This work was supported by the Natural Science Foundation of He’nan Province of China (112300410217).
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References [1] K.J. Huang, M. Zhang, W.Z. Xie, H.S. Zhang, Y.Q. Feng, H. Wang, Anal. Bioanal. Chem. 388 (2007) 939–946. [2] A.J. Gow, J.S. Stamler, Nature 391 (1998) 169–173. [3] J.S. Stamler, L. Jia, J.P. Eu, T.J. McMahon, I.T. Demchenko, J. Bonaventura, K. Gernert, C.A. Piantadosi, Science 276 (1997) 2034–2037. [4] A.R. Butler, P. Rhodes, Anal. Biochem. 249 (1997) 1–9. [5] C.M. Padget, Arch. Biochem. Biophys. 358 (1998) 232–242. [6] A. Samouilov, J.L. Zweier, Anal. Biochem. 258 (1998) 322–330. [7] A.S. Vidwans, S. Kims, D.O. Coffin, J. Neurochem. 72 (1999) 1843–1852. [8] A. Vliet, P.A.C. Hoen, P.S.Y. Wong, Am. Soc. Biochem. Mol. Biol. 273 (1998) 30255–30262. [9] K.J. Huang, W.Z. Xie, H. Wang, H.S. Zhang, Talanta 73 (2007) 62–67. [10] K.J. Huang, C.X. Xu, W.Z. Xie, H.S. Zhang, H. Wang, Spectrochim. Acta, Part A 69 (2008) 437–442. [11] L.P. Wang, H.X. Zhao, Y.M. Qiu, Z.Q. Zhou, J. Chromatogr. A 1136 (2006) 99– 105. [12] L.J. He, C.J. Wang, Y.J. Sun, X.L. Luo, J. Zhang, K. Lu, Int. J. Environ. Anal. Chem. 89 (2009) 439–448. [13] Q. Liu, J.B. Shi, L.X. Zeng, T. Wang, Y.Q. Cai, G.B. Jiang, J. Chromatogr. A 1218 (2011) 197–204.
[14] G. Basri, E. Yasun, M.I. Shukoor, Z. Zhu, M.X. You, X.H. Tan, H. Sanchez, D.H. Powell, H.J. Dai, W.H. Tan, J. Am. Chem. Soc. 132 (2010) 17408–17410. [15] L.A.L. Tang, J.Z. Wang, K.P. Loh, J. Am. Chem. Soc. 132 (2010) 10976–10977. [16] X.L. Dong, J.S. Cheng, J.H. Li, Y.S. Wang, Anal. Chem. 82 (2010) 6208–6214. [17] Y.B. Luo, Z.G. Shi, Q. Gao, Y.Q. Feng, J. Chromatogr. A 1218 (2011) 1353–1358. [18] C.L. Fu, W.S. Yang, X. Chen, D.G. Evans, Electrochem. Commun. 11 (2009) 997– 1000. [19] R. Muszynski, B. Seger, P.V. Kamat, J. Phys. Chem. C 112 (2008) 5263–5266. [20] Y.L. Yao, Y. Ding, L.S. Ye, X.H. Xia, Carbon 44 (2006) 61–66. [21] T. Kasuga, Thin Solid Films 496 (2006) 141–145. [22] J. Nawrocki, C. Dunlap, A. McCormick, P.W. Carr, J. Chromatogr. A 1028 (2004) 1–30. [23] M.H. Pinkse, P.M. Uitto, M.J. Hilhorst, B. Ooms, A.J.R. Heck, Anal. Chem. 76 (2004) 3935–3943. [24] C.T. Chen, Y.C. Chen, Anal. Chem. 77 (2005) 5912–5919. [25] K.J. Huang, H. Wang, M. Ma, H.S. Zhang, J. Chromatogr. A 1103 (2006) 193–201. [26] X. Zhang, H.Sh. Zhang, Spectrochimica 61 (2005) 1045–1049. [27] B. Saville, Analyst 83 (1958) 670–672. [28] K.J. Huang, Q.S. Jing, C.Y. Wei, Y.Y. Wu, Spectrochim. Acta, Part A 79 (2011) 1860–1865. [29] C.X. Xu, K.J. Huang, Y. Fan, Z.W. Wu, J. Li, J. Mol. Liq. 165 (2012) 32–37.
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction Ling-Ling Wang ⇑, Sheng Yu, Meng Yu College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" TiO2–Gr nanocomposite was used as
SPE adsorbent. " A novel spectrofluorimetry for
S-nitrosothiols determination was developed. " The developed method has low detection limit and wide linear range.
a r t i c l e
i n f o
Article history: Received 8 June 2012 Received in revised form 18 July 2012 Accepted 7 August 2012 Available online 23 August 2012 Keywords: S-nitrosothiols Spectrofluorimetric determination Solid-phase extraction TiO2–graphene nanocomposite
a b s t r a c t This present work describes the ultrasensitive and selective spectrofluorimetric determination of S-nitrosothiols by solid-phase extraction based on a novel adsorbent TiO2–graphene nanocomposite. 1,3,5,7Tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence is used as fluorescent probe for S-nitrosothiols label. The procedure is based on the fluorescent probe selective reaction with S-nitrosothiols to form highly fluorescent product, its extraction to the TiO2–graphene-packed SPE cartridge and spectrofluorimetric determination. The experimental variables affecting the extraction procedure, such as the type of the eluent and its volume, sample pH, and sample volume, have been studied. Under the optimized extraction conditions, the method showed good linearity in the range of 0.5– 100 nM. The limit of detection was 0.08 nM (signal-to-noise ratio = 3). Relative standard deviation was 2.5%. The developed method was applied to the determination of S-nitrosothiols in human blood samples with recoveries of 92.0–104.0%. This work revealed the great potentials of TiO2–graphene as an excellent sorbent material in the analysis of biological samples. Ó 2012 Elsevier B.V. All rights reserved.
Introduction The obligatory role of nitric oxide (NO) in the regulation of cardiovascular system has been widely recognized. However, NO is known to be extremely unstable and susceptible to inactivation by heme iron, non-heme iron, superoxide anion, oxygen, and other biochemical species [1]. Indeed, it has been suggested that NO could be stabilized by covalent bonding with thiols such as glutathione, cysteine, albumin, and hemoglobin [2,3], forming S-nitrosocysteine (SNOCys), S-nitrosoglutathione (SNOGSH), S-nitrosoalbumin, and ⇑ Corresponding author. Tel.: +86 371 6390811. E-mail address: [email protected] (L.-L. Wang). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.08.045
S-nitrosohemoglobin, respectively. Therefore, accurate evaluation of S-nitrosothiols (SNOs) in circulating blood is of great interest in assessing the in vivo regulatory state of NO/cGMP system not only by the endothelial NO, but also by NO-releasing vasodilators in clinical use such as glyceryl trinitrate [4]. Some analytical methods for the determination of RSNO have been developed. These mainly include spectrophotometry [5], chemiluminescence [6], electrometric methods [7], chromatography [8] and spectrofluorimetry [9,10], etc. However, the extremely low concentrations in biological samples and the complexity of their matrices make determination of SNOs challenging tasks. Because of this, there is a critical demand for rapid and simple preparation techniques especially extraction
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techniques, for evaluating and monitoring SNOs from different biological samples at trace level. Solid-phase extraction (SPE) is an efficient sample pre-treatment method, routinely used for the extraction of compounds from liquid or solid matrices. SPE approach, in which adsorbents have been treated as media for the retaining of target compounds, followed by selective elution, has been broadly utilized due to the advantages of less organic solvents, no emulsion formation like in liquid–liquid extraction, and short time, etc. [11]. The choice of appropriate adsorbent is a critical factor to obtain good recovery and high enrichment factor in SPE procedure. Graphene (Gr) is a fascinating carbon nanomaterial, which posses a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice. It has a large specific surface area (theoretical value 2630 m2/g) which suggests a high sorption capacity [12]. Furthermore, special structure makes both sides of the planar sheets of Gr are available for molecule adsorption. Actually, Gr and its composite have been reported using as adsorbent in SPE for different compounds enrichment [13–17]. However, many of the interesting and unique properties of Gr can only be realized after it is integrated into more complex assemblies [18]. Moreover, two-dimensional plane structure of Gr provides a vast platform for loading various nanoparticles, offering a new way to develop catalytic, magnetic, and optoelectronic materials [19]. It has also been reported that the integration of carbon-based materials and metal oxide usually shows synergistic effects in applications [20], so there is a reason to expect the integration of Gr and other nanoparticles has the similar effect on the SPE of SNOs. TiO2 nanoparticles is reported having large surface area and containing much ionexchangeable OH groups in the interlayer and surface [21], and it has gained great interests in analytical chemistry because of its high chemical stability, durability, corrosion resistance, non toxicity and cost effectiveness. Micro-scaled TiO2 has been used as stationary phase in HPLC applications [22] as well as a solid phase extraction adsorbent for selective extraction of phosphopeptides [23]. TiO2 were demonstrated to be excellent adsorbents of inorganic cations and anions as well as organic compound [24]. Spectrofluorimetric method of determination has several advantages including low detection limit, sensitivity, selectivity, low cost and less time consuming. Difluoroboradiaza-s-indacenes (BODIPY) has advantages of high extinction coefficients, high fluorescence quantum efficiency and stability to light [9]. It has been reported that Hg2+ can effectively displace NO from GSNO, and then NO reacts with o-phenylenediamine to form fluorescent derivative [9]. 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABO) is an excellent fluorescent probe [25]. Its self-fluorescence is very weak, and the quantum efficiencies can increase more than 100 times after reaction with NO. In this work, a novel method was developed for the ultrasensitive and selective determination of SNOs in biological samples using fluorescent probe 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABO) derivatization followed by SPE and spectrofluorimetric detection. TiO2–Gr was used as SPE adsorbents. Several key influence parameters were investigated in detail for good SPE efficiency. The method was demonstrated to be applicable for the analysis of SNOs in human blood samples. Experimental Apparatus All fluorescence measurements were performed on a Cary Eclipse Fluorescence Spectrophotometer (Varian, USA) with 1-cm
path length quartz cells. Instrument excitation and emission slits both were adjusted to 5 nm. The pH value of solution was measured using a pHS-3C meter (Shanghai Leici Equipment Factory, China). Scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 scanning electron microscope. The SPE cartridges (1 mL) were obtained from Agilent (Agilent, Palo Alto, CA, USA). Reagents 1,3,5,7-Tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)difluoroboradiaza-s-indacence (TMDCDABO) was synthesized according to the references [25,26]. The stock solution of 1 103 mol L1 TMDCDABO was prepared in methanol (MeOH). Glutathione (GSH) (reduced form, free acid 98–100%) was obtained from Shanghai Chemicals (Shanghai, China). Phosphate buffered saline (PBS) consisted of 10.0 g L1 NaCl, 0.25 g L1 KCl, 1.44 g L1 Na2HPO4, 0.25 g L1 KH2PO4 and adjusted to pH 7.0 [25]. Unless otherwise specified, all reagents were of analytical reagent grade used without further purification and all solutions were prepared from double-distilled water. Preparation of SNOs SNOs were prepared fresh for each experiment by incubating 100 mM thiol (GSH) with 100 mM sodium nitrite in the presence of 250 mM HCl and 0.1 mM EDTA for 30 min. The solutions rapidly turned red upon exposure to water, forming the corresponding SNOs. The stock solutions were stored on ice. The final concentrations were determined using the reported extinction coefficients at 334–338 nm for SNOGSH (e334nm = 780 mol1 cm1) [27]. Preparation of TiO2–Gr nanocomposite Graphene oxide (GO) was prepared from graphite powder by the modified Hummers method [28]. Graphite was added in a mixture containing 12 mL concentrated H2SO4, 2.5 g K2S2O8 and 2.5 g P2O5. The solution was heated to 80 °C and kept stirring for 5 h. The mixture was diluted with deionized water (500 mL). The product was obtained by filtering using 0.2 lm Nylon film and dried naturally. The product was re-oxidized by Hummers and Offeman method to produce the graphite oxide. Exfoliation was carried out by sonicating 0.1 mg mL1 graphite oxide dispersion for 1 h. TiO2– Gr nanocomposite was prepared according to the reference [29]. In short, 0.2 mL of Ti(OiPr)4 was added to the GO suspension (1 mg mL1) and ultrasonicated for 1 h. The mixture was then transferred to a 25-mL Teflon-sealed autoclave and kept at 130 °C for 12 h. The product was isolated by filtration and rinsed thoroughly with water and ethanol, respectively. The product was then dried in vacuum. The TiO2–Gr nanocomposite was obtained in the form of black powder. Derivatization procedures Hg2+ can effectively displace NO from GSNO, and then NO reacts with TMDCDABO to form TMDCDABO derivative. The derivatization procedures were according to the reference [10]. 2 mL 2.0 105 mol L1 TMDCDABO was transferred to a 10 mL volumetric flask. Then 2 mL PBS, 0.05 mL 5 104 mol L1 SNOGSH and 1 mL of 1 103 mol L1 Hg2+ solution were added. The mixture was diluted to the mark with water and kept at 30 °C for 15 min. The chemical structure of TMDCDABO and its reaction with SNOGSH are shown in Fig. 1.
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NH2
NH2 O
O OH N S
NH H N
O
O
O
O Hg2+
O OH
OH HS
NH H N
O
+
NO
OH O
NH2
HN N N
NH2 H3C
339
CH3
CO2C2H5 N N B H3C F F CH3
NO
H3C
CH3
C2H5O2C
C2H5O2C
O2
CO2C2H5 N N B H3C F F CH3
Fig. 1. The reaction of TMDCDABO with SNOGSH.
Solid phase extraction procedure and spectrofluorimetric analysis TiO2–Gr (20 mg) was added in a 1 mL SPE cartridge using an upper frit and a lower frit to avoid adsorbent loss. Prior to extraction, the cartridge was preconditioned with 5 mL MeOH and 5 mL water successively. The sample solution (10 mL) was then passed through the cartridge. The analytes retained on the cartridge were subsequently eluted with 1 mL MeOH. The eluate was collected and the fluorescence was measured at kex/kem = 500/510 nm against a reagent blank carried through the same procedure except for the addition of SNOGSH.
recovery was defined as the percentage of total analyte which was extracted to SPE column.
Extraction recovery ¼ ½ðC 0 V 0 Þ ðC 1 V 1 Þ 100=ðC 0 V 0 Þ ð1Þ where C1 and C0 are the concentration of the analyte in the final and the initial concentration in the sample, respectively. V1 and V0 are the volumes of the samples involved.
Results and discussion Samples preparation
Morphology of graphene
Human blood samples were collected from free volunteers. All samples were obtained by venipuncture using sodium citrate as an anticoagulant and kept on ice in the dark. After bubbling oxygen for 10 min, the derivatization procedures were performed as described above. Subsequently, the mixture was centrifuged for 10 min at 3000 rpm and the supernatant was collected and determined by SPE-spectrofluorimetry method.
The morphology of the obtained Gr and TiO2–Gr was observed by SEM (Fig. 2). Fig. 2A shows the SEM image of Gr, revealing the typical crumpled and wrinkled Gr sheet structure. Fig. 2B presents the SEM image of TiO2–Gr composite. It shows the TiO2 nanoparticles are uniformly and compactly embedded on the Gr substrate.
Extraction recovery
In order to obtain the maximal extraction efficiency, several factors such as the elution solvent, the sample solution volume and pH value, flow rate of sample solution were optimized. All the optimization experiments were conducted three times.
One main parameter has been employed for the evaluation of the proposed configuration, namely: extraction recovery. Extraction
Optimization of the solid phase extraction procedure
Fig. 2. SEM image of Gr (A) and TiO2–Gr (B).
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120
80
Recovery (%)
Recovery (%)
100
60 40
A
100
80
60
20 40
0 Hexane
DCM
ACN
MeOH
0.5
1.0
Elution solvent
1.5
2.0
2.5
3.0
Elution volume (mL)
Fig. 3. Effect of elution solvent on recoveries. Eluent volume: 1 mL; sample flow rate: 2 mL min1; sample pH 7.0; sample volume: 10 mL.
110
B
Effect of flow rate of sample solution through the cartridge Flow rate of aqueous sample solution always has a significant impact in the SPE procedure, because the sample flow rate affects both the recoveries of analytes and loading time in a SPE system. The influence of sample flow rate was investigated over the range of 1–5 mL min1 with the other conditions kept constant. Fig. 4B shows that 2 mL min1 is optimal flow rate which is selected for further use in the following experiment. Effect of sample pH Sample pH plays an important role in the SPE procedure and affects the extraction efficiency. pH factor determine the surface charge of the TiO2–Gr nanoparticles. The –OH and –OOH groups can be protonated in acidic solution then TiO2–Gr has positive surface charge. However, in alkaline media the –OH and –OOH groups can be deprotonated and they have negative surface charge. The effect of sample pH on the recoveries of SNOGSH derivative was examined in a range of 4–9. The highest recovery is obtained at pH 7 (Fig. 4C). The analyte SNOGSH derivative is electric neutrality,
Recovery (%)
100 90 80 70 60 50 1
2
3
4
5 -1
Flow rate (mL min ) 110
C
100
Recovery (%)
Effect of elution solvent The type of elution solvent is important for the extraction efficiency [28]. The TiO2–Gr-packed cartridges were first washed thoroughly with water and various organic solvents including MeOH, acetonitrile (ACN), tetrahydrofuran (THF), and hexane, ethanol, acetone, dichloromethane. No visible adsorbent losses were observed. Four solvents with different polarities were tested: hexane, dichloromethane (DCM), acetonitrile (ACN) and MeOH. As shown in Fig. 3, MeOH yields the higher recovery (97.2%) than that of ACN (82.3%), because protic solvent (i.e., MeOH) can elute the polar derivative that may have hydrogen bonding with the hydroxyl groups on the TiO2–Gr surface more effectively than aprotic solvent (i.e., ACN). DCM also cannot satisfactorily elute the analyte with the recovery of 78.1%. Hexane has a poor eluting ability toward SNOGSH derivative (recovery 45.4%). Therefore, MeOH was selected for elution of analytes from the TiO2–Gr-packed SPE cartridges. The volume of the eluent has a great effect on the elution performance and efficiency. To find out the required MeOH volume to recover all the analytes from TiO2–Gr packed cartridge, eluent volumes in the range of 0.5–3 mL were tested. From Fig. 4A, it suggested that SNOGSH derivative needed a volume of eluent more than 1 mL to get the best recoveries. Thus, to achieve complete elution of the seven analytes, 1 mL MeOH was utilized in following experiments.
90 80 70 60 50 40 4
5
6
7
8
9
pH Fig. 4. Effects of elution volume (A), sample flow rate (B) and sample pH (C) on the recoveries. Other conditions are the same in Fig. 3.
and the most SNOGSH derivative is adsorbed on the SPE adsorbent at pH 7. So pH 7 was chosen as the pH of the sample solutions. Effect of sample volume To obtain reliable analytical results and high enrichment efficiency, it was necessary to obtain the breakthrough volumes in the SPE process. Different volumes (1, 3, 5, 10, 20 mL) of samples were investigated three times for the breakthrough volume. The results showed that the breakthrough volume of SNOGSH derivative was more than 20 mL at least. In practice, the sample volume was chosen according to the required sensitivity and the time acceptable for a whole analysis. Generally, further increasing of
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the sample volume was not desirable for routine analysis since the total time needed for one analysis would be longer. So a sample volume of 10 mL was selected for subsequent analysis with satisfactory sensitivity. Effect of amount of cartridge packing The efficiency of the TiO2–Gr amount on the recovery of analytes was studied. Four solid phase amounts were tested (5, 10, 20 and 30 mg) for the preconcentration of SNOGSH derivative. Obtained results showed insignificant differences in recoveries among the different amounts of cartridge packing. The highest recovery was obtained when 20 mg TiO2–Gr was used. Therefore, the TiO2–Gr amount of 20 mg was recommended for the extraction of SNOGSH derivative.
Table 1 Analytical characteristics of the developed method. Analytical parameters
Without SPE
With SPE
Linear range (nM) Regression equation Detection limit (nM) Correlation coefficient (R) Relative standard deviation (n = 6)
10–120 Y = 2.56 X + 20.439 1.0 0.9994 1.8
0.5–100 Y = 8.20 X + 45.21 0.08 0.9992 2.5
Excitation and emission slits both were 5 nm; kex/kem = 500/510 nm.
Table 2 Analytical results of the blood samples (n = 6). Samples
Added (nM)
Found (nM)
Recovery
RSD (%)
1
0 1 10 60 0 1 10 60 0 1 10 60
9.26 10.18 18.94 67.58 10.12 11.14 19.68 67.90 9.75 10.79 19.57 71.19
– 92.0 96.8 97.2 – 102.0 95.6 96.3 – 104.0 98.2 102.4
2.8 1.6 3.2 2.4 4.1 4.3 3.2 3.1 2.8 2.6 3.6 3.2
Comparison study The present results with TiO2–Gr, as new sorbent for the SPE, was compared to Gr and traditional method using C18 silica sorbent. SNOGSH derivative were extracted from by Gr and C18 procedure as the same as the TiO2–Gr. Fig. 5 shows that the extraction recovery of TiO2–Gr is the highest than that of Gr and C18. Although the recoveries obtained by TiO2–Gr was close to that by Gr, the elution of the analytes from the Gr-packed SPE cartridge consumed more time than from TiO2–Gr-packed SPE cartridge. The reason may be Gr is prone to congregate, and this hinders the objective molecules elution. Furthermore, it is necessary to take into account the cost of adsorbent. C18 silica sorbents are more expensive than the TiO2–Gr laborated with the simple hydrothermally method. Method validation To validate the applicability of the proposed SPE method using the TiO2–Gr sorbent, linearity and limit of detection (LOD) were assessed using the optimum extraction conditions. Table 1 shows that good linearity is obtained for SPE extractions using the TiO2–Gr sorbent with coefficient of determination (R = 0.9992). The LOD is 0.08 nM, which is calculated at a signal to noise ratio (S/N) of 3. The repeatability (RSD) is 2.5%. These values were better than that for the spectrofluorimetry without SPE procedure (Table 1). The TiO2–Gr cartridge was found practically reusable for all the extractions performed. The potential regeneration and stability of the TiO2–Gr cartridge were studied up to at least 30 adsorption-elution cycles. The column was washed with 5 mL of MeOH followed by 5 mL deionized water after each extraction. It was observed that TiO2–Gr SPE could
3
Elution solvent: MeOH; eluent volume: 1 mL; sample flow rate: 2 mL min1; sample pH 7.0; sample volume: 10 mL; excitation and emission slits both were 5 nm; kex/kem = 500/510 nm.
be repeatedly used (up to at least 30 adsorption-elution cycles) without significant loss of uptake capacity in the extraction of SNOGSH derivative. The interday precisions (RSDs) after 25 cycles of use were 3.2%. Furthermore, the cost of producing the TiO2–Gr was low and the process involve was simple and easily performed. Sample analysis The proposed method was applied to the determination of trace-level S-nitrosothiols in the blood samples collected from free volunteers. All samples were analyzed for six times using the same recommended procedure and conditions. The results are shown in Table 2. The recoveries ranged from 92.0% to 104.0% and the RSD from 1.6% to 4.3%, which confirmed the feasibility and reliability of the present method. Conclusions TiO2–Gr has been successfully applied as sorbent for SPE coupled with spectrofluorimetry for the determination of S-nitrosothiols from blood samples. LOD at about part per trillion level Snitrosothiols in blood samples was achieved using this nanocomposite. The SPE method presented here offers an interesting and effective option for the analysis of S-nitrosothiols and other spectrofluorimetry-amenable trace biomoleculars in similar sample types or similar analytes from other sample types. A comparison of TiO2–Gr SPE with commercial C18 SPE and Gr SPE indicates that TiO2–Gr sorbent is better in terms of recovery. TiO2–Gr sorbent is also cost effective and easy to prepare. The high surface area of the material facilitate in the adsorption process and its increase in sensitivity.
100
Recovery (%)
2
80 60 40 20 0 TiO2-Gr
Gr
C18
Fig. 5. Effect of adsorbent type on recoveries. Other conditions are the same in Fig. 3.
Acknowledgments This work was supported by the Natural Science Foundation of He’nan Province of China (112300410217).
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References [1] K.J. Huang, M. Zhang, W.Z. Xie, H.S. Zhang, Y.Q. Feng, H. Wang, Anal. Bioanal. Chem. 388 (2007) 939–946. [2] A.J. Gow, J.S. Stamler, Nature 391 (1998) 169–173. [3] J.S. Stamler, L. Jia, J.P. Eu, T.J. McMahon, I.T. Demchenko, J. Bonaventura, K. Gernert, C.A. Piantadosi, Science 276 (1997) 2034–2037. [4] A.R. Butler, P. Rhodes, Anal. Biochem. 249 (1997) 1–9. [5] C.M. Padget, Arch. Biochem. Biophys. 358 (1998) 232–242. [6] A. Samouilov, J.L. Zweier, Anal. Biochem. 258 (1998) 322–330. [7] A.S. Vidwans, S. Kims, D.O. Coffin, J. Neurochem. 72 (1999) 1843–1852. [8] A. Vliet, P.A.C. Hoen, P.S.Y. Wong, Am. Soc. Biochem. Mol. Biol. 273 (1998) 30255–30262. [9] K.J. Huang, W.Z. Xie, H. Wang, H.S. Zhang, Talanta 73 (2007) 62–67. [10] K.J. Huang, C.X. Xu, W.Z. Xie, H.S. Zhang, H. Wang, Spectrochim. Acta, Part A 69 (2008) 437–442. [11] L.P. Wang, H.X. Zhao, Y.M. Qiu, Z.Q. Zhou, J. Chromatogr. A 1136 (2006) 99– 105. [12] L.J. He, C.J. Wang, Y.J. Sun, X.L. Luo, J. Zhang, K. Lu, Int. J. Environ. Anal. Chem. 89 (2009) 439–448. [13] Q. Liu, J.B. Shi, L.X. Zeng, T. Wang, Y.Q. Cai, G.B. Jiang, J. Chromatogr. A 1218 (2011) 197–204.
[14] G. Basri, E. Yasun, M.I. Shukoor, Z. Zhu, M.X. You, X.H. Tan, H. Sanchez, D.H. Powell, H.J. Dai, W.H. Tan, J. Am. Chem. Soc. 132 (2010) 17408–17410. [15] L.A.L. Tang, J.Z. Wang, K.P. Loh, J. Am. Chem. Soc. 132 (2010) 10976–10977. [16] X.L. Dong, J.S. Cheng, J.H. Li, Y.S. Wang, Anal. Chem. 82 (2010) 6208–6214. [17] Y.B. Luo, Z.G. Shi, Q. Gao, Y.Q. Feng, J. Chromatogr. A 1218 (2011) 1353–1358. [18] C.L. Fu, W.S. Yang, X. Chen, D.G. Evans, Electrochem. Commun. 11 (2009) 997– 1000. [19] R. Muszynski, B. Seger, P.V. Kamat, J. Phys. Chem. C 112 (2008) 5263–5266. [20] Y.L. Yao, Y. Ding, L.S. Ye, X.H. Xia, Carbon 44 (2006) 61–66. [21] T. Kasuga, Thin Solid Films 496 (2006) 141–145. [22] J. Nawrocki, C. Dunlap, A. McCormick, P.W. Carr, J. Chromatogr. A 1028 (2004) 1–30. [23] M.H. Pinkse, P.M. Uitto, M.J. Hilhorst, B. Ooms, A.J.R. Heck, Anal. Chem. 76 (2004) 3935–3943. [24] C.T. Chen, Y.C. Chen, Anal. Chem. 77 (2005) 5912–5919. [25] K.J. Huang, H. Wang, M. Ma, H.S. Zhang, J. Chromatogr. A 1103 (2006) 193–201. [26] X. Zhang, H.Sh. Zhang, Spectrochimica 61 (2005) 1045–1049. [27] B. Saville, Analyst 83 (1958) 670–672. [28] K.J. Huang, Q.S. Jing, C.Y. Wei, Y.Y. Wu, Spectrochim. Acta, Part A 79 (2011) 1860–1865. [29] C.X. Xu, K.J. Huang, Y. Fan, Z.W. Wu, J. Li, J. Mol. Liq. 165 (2012) 32–37.