Analytica Chimica Acta 816 (2014) 41–49
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A surface-enhanced Raman scattering method for detection of trace glutathione on the basis of immobilized silver nanoparticles and crystal violet probe Lei Ouyang a,b , Lihua Zhu a,b , Jizhou Jiang a , Heqing Tang b,∗ a
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P.R. China Key Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission and Ministry of Education, College of Chemistry and Materials Science, South Central University for Nationalities, Wuhan 430074, P.R. China b
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
• A SERS substrate was synthesized by fixing Ag onto Fe3 O4 magnetic particles. • The analysis of Raman insensitive GSH was achieved with Raman sensitive crystal violet as a probe. • The new method can determine GSH in the range of 50–700 nmol L−1 with a detection limit of 40 nmol L−1 . • The proposed method has good selectivity and reproducibility for trace GSH detection in real samples.
a r t i c l e
i n f o
Article history: Received 18 September 2013 Received in revised form 19 January 2014 Accepted 24 January 2014 Available online 3 February 2014 Keywords: Surface-enhanced Raman scattering Glutathione Immobilized silver nanoparticles Crystal violet Replacement.
a b s t r a c t Unsatisfactory sensitivity and stability for molecules with low polarizability is still a problem limiting the practical applications of surface-enhanced Raman scattering (SERS) technique. By preparing immobilized silver nanoparticles (Fe3 O4 /Ag) through depositing silver on the surface of magnetite particles, a highly sensitive and selective SERS method for the detection of trace glutathione (GSH) was proposed on the basis of a system of Fe3 O4 /Ag nanoparticles and crystal violet (CV), in which the target GSH competed with the CV probe for the adsorption on the Fe3 O4 /Ag nanoparticles. Raman insensitive GSH replaced the highly Raman sensitive CV adsorbed on the surface of Fe3 O4 /Ag particles. This replacement led to a strong decrease of the CV SERS signal, which was used to determine the concentration of GSH. Under optimal conditions, a linear response was established between the intensity decrease of the CV SERS signal and the GSH concentration in the range of 50–700 nmol L−1 with a detection limit of 40 nmol L−1 . The use of a Fe3 O4 /Ag substrate provided not only a great SERS enhancement but also a good stability, which guarantees the reproducibility of the proposed method. Its use for the determination of GSH in practical blood samples and cell extract yielded satisfactory results. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +86 27 67843323; fax: +86 27 67843323. E-mail addresses: [email protected], [email protected] (H. Tang). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.046
Glutathione (␥-L-glutamyl-L-cysteinyl-glycine; GSH) is the most abundant nonprotein thiol source in mammalian tissues and plays a pivotal role in biological processes [1]. It has both reduced form (GSH) and oxidized dimeric form (GSSG). By the conversion of the two forms, it can maintain thiol-disulfide reduction–oxidation potential. It also performs as an important
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intracellular reduction–oxidation buffer, which may trap free radicals and peroxides to prevent cell damage [2]. The ratio of GSH/GSSG reflects the ability of antioxidant and is able to be used for monitoring the cells overall health. GSH has many other biological functions like detoxication, signal transduction, and transportation/elimination of metal ions since thiol is involved in most of biological processes [3]. Medical studies show that the level of GSH in plasma is related to diseases such as HIV, Alzheimer’s disease, and Parkinson’s disease [4]. Hence it is needed to develop sensitive and convenient methods for the detection of GSH in biological matrix. The detection of GSH and other thiols can be realized by using enzymatic [5], florescence (FL) [6,7], mass spectrometric (MS) methods [8]. The separation methods using liquid chromatography (LC) and capillary eletrophoresis (CE) are most popular for selective and sensitive determinations of GSH, giving the methods such as LC-chemiluminiscence (LC-CL) [9], LC-florescence (LC-FL) [10], CE-laser induced FL [11]. Although a number of techniques have been employed to assay the thiols, some problems still occur due to the operation of sophisticated instrumentation and/or the complexity of the procedure. Another strategy for the detection of GSH without separation is feasible by taking account of its reactive sulfydryl (–SH) group. This group has a strong interaction with many metal ions such as Hg, Au and Ag [12] and many organofunctional groups such as aldehyde group. These interactions are used to develop CL [13], electrochemical [14] and colorimetric [15] methods for quantifying GSH. Wang et al. synthesized grapheneCdTe composite with a selective combination to –SH, and used it as an electrochemical probe for GSH detection [16]. By using a fluorescence probe containing Tb(III), McMahon et al. determined the concentration of GSH [17]. Han et al. prepared Hg-modified CdTe quantum dot with a high sensitivity to biological thiol [18]. Taking account of the different association constant of DNA and GSH with Hg ions, Xu et al. detected GSH by using the replacement reaction between DNA and GSH [19]. Since the environmental risk of heavy metals, some heavy-metal-free probes have also been proposed. Tanaka et al. used an aldehyde to react with GSH, and used the induced fluorescence decrease to detect the concentration of GSH [20]. However, these probes for GSH usually consisted of short wavelength emitting and short lifetime-based fluorophores, which showed many drawbacks when used in biologic media. Surface-enhanced Raman scattering (SERS) is a sensitive analytical technique [21,22], and it is especially advantageous for treating biological samples because it can analyze aqueous samples without disturbance of water [23,24]. SERS should have a prospect in GSH detection. However, due to the low polarization of GSH, its direct SERS detection is not sensitive. Larsson et al. reported that the SERS detection of GSH on nano silver had a limit of detection as high as 0.5 mmol L−1 [25]. A heat-induced SERS method was reported to detect low level GSH, but suffering strong interferences [26]. A Raman-probe mediated method is helpful to overcome the difficulty from low polarizing molecules [27,28]. On a nano silver substrate, Huang et al. used rhodamine B as probe for the detection of GSH and made a great achievement by decreasing the detect limit to 1 mol L−1 GSH [29]. However, according to our experiences, simple nano silver gel is not a good SERS substrate for liquid detection, because the nano particles exhibit low uniformity and low stability, being unfavorable to the reproducibility of the analytical method [30]. Therefore, it is needed to develop better SERS substrate. Silver- and gold-coated iron oxides were recently reported as SERS substrate due to their wide range of potential uses in industrial, biomedical, and environmental applications [31,32]. Du et al. prepared Fe3 O4 /Ag as a substrate for the detection of PAHs and Cr(VI) [33,34]. Zhou et al. synthesized core-shell Fe3 O4 /Au for the protein detection [35]. Shen et al. used Fe3 O4 /Ag/SiO2 /Au
as substrate for the study of long-range plasmon coupling effect on the SERS responses [36]. These nanocomposites combined the advantages of “core” and “shell”, providing such composites with enhanced stabilization as well as SERS effect and greatly extending the application of such substrate. When nanocomposites are used as a SERS substrate, their uniform particle size distribution is very important for the reproducibility of the SERS method. In the present work, we tried to develop a better substrate and find a better Raman probe for sensitive and indirect detection of GSH. In consideration of the reported methods of nano gold/silver fixing and our experiences in the ultrasonic preparation of Fe3 O4 particles [37], we prepared Fe3 O4 particles with a narrow particle size distribution, and then synthesized Fe3 O4 /Ag as a SERS substrate by in-situ reduction of silver ions. Fe3 O4 /Ag particles are easily magnetized by an external magnetic field, being favorable to the localization when they serve as an efficient SERS substrate. We chose Raman sensitive molecule crystal violet (CV) as a Raman probe. The purple dye CV can easily adsorb to the noble metal substrate and has a maximum absorption wavelength around 591 nm, which matches with the laser wavelength of 532 nm being used for the SERS measurement. These properties of CV make it a good Raman probe in SERS [38,39]. By combining these two selections (the Fe3 O4 /Ag substrate and CV probe), a sensitive SERS method was developed for analyzing GSH with good reproducibility and anti-interference ability. 2. Experimental 2.1. Reagents Ferric chloride (FeCl3 ), ferrous sulfate (FeSO4 ), silver nitrate (AgNO3 , >99.8%), sodium hydroxide (NaBH4 , >99.5%), aqueous ammonia (25%), sodium chloride, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium hydrogen carbonate, sodium nitrate, sodium chloride, calcium nitrate tetrahydrate, zinc nitrate hexahydrate, potassium nitrate, trichloroacetic acid (TCA), CV and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd (China). 3Aminopropyltrimethoxysilane (APTMS, 97%), cystine (Cyss), GSSG, N-acetylcysteine (NAC) was from Aladdin Industrial Corporation (USA). GSH was from Bio Basic Inc. (Canada). Cysteine (Cys), glycline (Gly), serine (Ser), threonine (Thr), glutamine (Glu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) were obtained from Shanghai Ruji Biological Technology Development Co., Ltd. (China). All reagents were of analytical reagent grade and used without further purification. 2.2. Preparation of Fe3 O4 MNPs Fe3 O4 nanoparticles were synthesized by an ultrasonic assisted reverse co-precipitation method [37]. In brief, 5.0 mL of 1.0 mol L−1 FeCl3 solution and 10.0 mL of 0.5 mol L−1 FeSO4 solutions were first mixed and then added dropwise into 20 mL of 3.5 mol L−1 ammonia water at 60 ◦ C with ultrasound irradiation for 30 min which was carried out in an ultrasound clean bath operating at 25 kHz with a power of 140 W (KQ-200KDE, China). The produced black Fe3 O4 nanoparticles were collected by magnetic separation, washed with water to neutral pH, and dried in a vacuum oven at 60 ◦ C. 2.3. Preparation of Fe3 O4 /Ag particles Fe3 O4 (0.05 g) was dispersed in 20 mL ethanol and sonicated for 5 min. After the addition of 200 L APTMS, the solution was vigorously mixed for 6 h. The APTMS modified Fe3 O4 particles were separated by an applied magnetic field and washed with ethanol and water. The next procedure was in-situ reduction Ag+ to Ag. The
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APTMS-coated Fe3 O4 (0.05 g) was dispersed in 20 mL of 0.1 mol L−1 AgNO3 , and the reaction was initiated by adding 20 mL 0.2 mol L−1 NaBH4 dropwise into the solution with stirring, being followed by further stirring for 45 min. The particles were then obtained with an applied magnetic field and washed fully with water. Finally, the obtained Fe3 O4 /Ag NPs were re-dispersed in 50 mL of water.
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3. Result and discussion 3.1. Substrate characterization
A GSH stock solution of 0.01 mol L−1 was prepared by dissolving GSH into diluted water. The Fe3 O4 /Ag dispersion was pre-equilibrated with an aqueous solution of the probe CV by mixing them before the detection of GSH and stirring for 15 min. Then the GSH sample solution was added to the CV solution, solution pH was adjusted to 6.5 with NaOH solution and the co-adsorption was conducted for another 20 min. The NPs were assembled by using an applied magnetic field for SERS detection. Part of the particles with solution (10 L) was taken out for SERS detection. All SERS and conventional Raman spectra were measured with a DXR Raman Microscopy equipped with a CCD detector (Thermo Fisher Scientific, USA). The 532 nm line of NIR laser was used as an excitation source. The laser power was 3.0 mW at the sample position and the exposure time for each SERS measurement was set at 2 s with 2 accumulations in this study.
As shown in Fig. 1a, there were a series of characteristic peaks at 2 = 30.4◦ , 35.7◦ , 43.5◦ , 53.9◦ , 57.15◦ and 63.1◦ in the patterns of both Fe3 O4 and Fe3 O4 /Ag, which fit well with XRD PDF card 750449 of Fe3 O4 . Unlike Fe3 O4 , the Fe3 O4 /Ag sample also gave a series of peaks at 2 = 38.2◦ , 44.4◦ , 64.6◦ and 77.6◦ , which are the characteristic peaks of Ag according to XRD PDF card 87-0720. This signs the deposition of Ag on the surface of Fe3 O4 MNPs. The average core size of the particles was evaluated with the Debye–Sherrer formula D = K/(ˇ cos), where K is the Sherrer constant (0.89), is the X-ray wavelength (0.15418 nm), ˇ is the peak full width at half maximum (FWHM), and is the Bragg diffraction angle. From the peak (3 3 1) of Fe3 O4 and (1 1 1) of Ag, the average crystal size of Fe3 O4 and Fe3 O4 /Ag were calculated to be 16.22 and 24.18 nm, respectively. XPS was performed to provide elemental information of the surface atomic composition of Fe3 O4 /Ag. Fig. 1b exhibited the XPS spectrum of Fe3 O4 /Ag, which clearly showed the signals for Fe, Ag, O, and C element, the atomic percentage of each element was 25.82%, 5.12%, 50.29% and 18.77%. The XPS spectrum of Fe (2p) and Ag (3d) were shown in Fig. 1c and d. The Fe 2p1/2 and 2p3/2 peaks were centered at 724.2 eV and 710.8 eV, respectively, which were characteristics of Fe3 O4 [37]. This suggests that no significant change in peak position was observed upon the coating of Ag. The Fe (2p) spectra were deconvoluted by taking into account contributions from Fe(II), Fe(III) and the satellite peak. The Fe (2p) peaks at binding energies of 711.2 eV and 725.4 eV with a satellite signal at 718.2 eV were characteristic of Fe(III), while the peaks of 709.9 eV and 723.4 eV with a satellite signal at 713.4 eV were characteristic of Fe(II). The atomic ratio of Fe(III) to Fe(II) was about 2:1, confirming the formation of Fe3 O4 . In the envelop of Ag (3d), two peaks located at 367.6 and 373.6 eV with a spin-orbit splitting of 6 eV, showing the formation of Ag layer [40]. Both the XRD and XPS results demonstrate that metallic of Ag was coated on the Fe3 O4 surface. On a laser scattering particle size distribution analyzer, the average sizes of Fe3 O4 and Fe3 O4 /Ag particles were measured to be 345 nm and 260 nm, respectively. This hints that the deposition of Ag is favorable to depressing the aggregation of Fe3 O4 particles, possibly due to the capping effect of the introduced APTMS for the deposition of Ag.
2.6. Extract collection and analysis of whole blood samples and hepatoma cells
3.2. Displacement effect of GSH on Raman responses of CV and the possible mechanism
The method of McDermott et al. [9] was followed for collecting and processing blood samples. The blood samples (3 mL) were drawn from several healthy adult volunteers and put into the blood tubes immediately, then reserved at 0–4 ◦ C. Noticing that the perchloric acid may oxidize GSH during the adsorption process, we used TCA instead of perchloric acid, which was used in the method of McDermott et al. [9]. For whole blood analysis, 500 L of blood was added into the eppendorf tube with 500 L TCA (10%, w/v), the tubes were then vortexed and incubated on ice for 10 min. After the blood proteins were precipitated, the suspensions were then centrifuged at 14,000 rpm for 5 min, and the supernatant was diluted and analysed with the proposed method. Hepatoma cell HepG2 and Be17402 were collected after trypsinization and washed with phosphate buffer (PBS) (pH 7.4). After counting, the cells were centrifuged. The precipitated pellet was mixed with 10% TCA and reacted for 6 h with stirring. Then the cells were centrifuged, the supernatant was collected. The pellet was washed with TCA and the supernatant was mixed with the former supernatant for GSH detection.
CV is a common cationic dye with a symmetry molecule structure. The solution of CV at a concentration of 1 mg L−1 (2.4 mol L−1 ) yielded very weak Raman response (curve 1 in Fig. 2a). By using the Fe3 O4 /Ag substrate (load 25 mg L−1 ), the Raman signal of CV was enhanced by a factor of about 105 (curve 2 in Fig. 2a), demonstrating that the Fe3 O4 /Ag particles are an excellent SERS substrate and CV is a good Raman probe. After the addition 10 mol L−1 of GSH, the SERS intensity of CV on the Fe3 O4 /Ag substrate was decreased significantly by a factor of 10 (curve 3 in Fig. 2a). The significant displacement effect of GSH on the SERS responses of CV was attributed to the fact that less Raman active GSH adsorbs more strongly on the surface of the Fe3 O4 /Ag substrate than CV does, as discussed below. The adsorption competition between GSH and CV was confirmed by comparing the related UV-vis absorption spectra. As shown in Fig. 2b, CV (2 mg L−1 ) had a strong absorption band with the maximum absorption wavelength at 591 nm (curve 1). After CV (2 mg L−1 ) was mixed with Fe3 O4 /Ag (load 125 mg L−1 ) for 15 min, the UV-vis absorption spectrum of the residual solution
2.4. Characterization X-ray diffraction (XRD) patterns were recorded on an X’Pert PRO X-ray diffractometer (PANalytica) with a Cu K␣ radiation source generated at 40 kV and 30 mA. The particle size distribution was measured by a dynamic light-scattering method on a ZEN 3690 Zetasizer (Malvern, United Kingdom). The surface chemical compositions of Fe3 O4 /Ag MNPs were identified by X-ray photoelectron spectroscopy (XPS, XSAM 800, KROTOS) using the Mg K␣ line (1253.6 eV) as an excitation source. The pressure in the XPS analysis chamber was maintained at 10−9 mbar. The binding energies of all peaks were referenced to the C (1s) line (285.0 eV). Before the XRD and XPS measurements, the prepared Fe3 O4 /Ag nanoparticles were collected by magnetic separation and vacuum dried at 50 ◦ C for 1 h. Such a pre-treatment could not cause the change of the crystalline structure and the oxidation of metallic silver in the Fe3 O4 /Ag samples. 2.5. SERS analysis
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Fig. 1. (a) XRD patterns of Fe3 O4 and Fe3 O4 /Ag. (b) XPS survey scan spectrum, (c) Fe 2p envelop and (d) Ag 3d envelop of Fe3 O4 /Ag.
was recorded after the solid particles were removed off (curve 2). In comparison with the CV solution, the absorbance of the residual solution was decreased, indicating that the CV concentration in the residual solution was decreased due to its adsorption on the substrate. In the third run, 0.1 mmol L−1 of GSH was mixed with the mixture of CV and Fe3 O4 /Ag, and then the UV-vis absorption spectrum of the second residual solution was recorded (curve 3) after the solid particles were removed off. By comparing curves 2 and 3 in Fig. 2b, it was found that the CV concentration was recovered to an extent in the second residual solution. This hinted that parts of the adsorbed CV molecules were released from the substrate surface due to the stronger adsorption of GSH. To make clear the mechanism of the replacement, we made a comparison between SERS spectra of Fe3 O4 /Ag and GSH (0.5 mol L−1 ) with the Raman spectrum of solid GSH. As shown in Fig. 3, in the Raman spectrum of solid GSH, there was a characteristic peak at 2571 cm−1 , corresponding to the stretch vibration of–SH. This peak disappeared in the SERS spectrum of and GSH on Fe3 O4 /Ag, indicating that the –SH group was eliminated by the adsorption of GSH on the surface of the substrate. At the same time, a new peak appeared at 236 cm−1 in the SERS spectrum of GSH, which corresponded to the Ag-S bond. This supported that the adsorption of GSH on the Fe3 O4 /Ag was achieved by forming Ag-S bond. The mechanism of Ag-S bond formation was further clarified by using SCN− (25 mmol L−1 ) as a probe. Since the stretch vibration of
C N (CN ) is very sensitive to the coordination environment, the electron transformation of targeting molecule could be studied by observing the variations in CN [41]. As shown in Fig. 4, the characteristic peak of CN appeared at 2117 cm−1 , but the addition of GSH made the peak red shift. When the GSH concentration was increased to 10−3 mol L−1 , the red shift was increased to 20 cm−1 . This phenomenon could be explained in terms of the electrondonor effect of GSH: The S atom of the adsorbed GSH donated its lone pair electrons to silver surface, and then the negative charge on the silver surface transferred to SCN− through the generated S-Ag bond. The conjugated effect between S atom and C N bond made it possible to increase the electron density of C N bond and consequently weaken the C N bond and decreased the frequency of C N. This GSH-induced red shift of CN confirmed again that the GSH interacted with Fe3 O4 /Ag by forming Ag-S through the donation effect of the S atom in the GSH molecule. Since the Ag-N bond is weaker than the Ag-S bond [42], the replacement of Ag-N by Ag-S could take place, which led to the decrease of SERS signals of CV. This displacement effect could be used to detect GSH indirectly. 3.3. Effect of CV concentration and adsorption time To optimize the concentration of the probe CV, the concentration dependence of its SERS intensity at 1620 cm−1 was investigated with a load of 25 mg L−1 Fe3 O4 /Ag as the substrate. As shown in Fig. 5a, the SERS intensity was increased with
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Fig. 4. SERS spectra of SCN− (0.025 mol L−1 ) in presence different concentrations of GSH.
Fig. 2. (a) A comparison between (1) Raman spectrum of CV, (2) SERS spectrum of CV and (3) SERS spectrum of CV in the presence of 10 mol L−1 GSH. Other experimental conditions: substrate Fe3 O4 /Ag 25 mg L−1 ; CV concentration 1 mg L−1 ; CV pre-adsorption time 15 min; GSH adsorption time 20 min. (b) UV-vis absorption spectra of the residual CV in solution (particles have been removed): (1) CV, (2) CV + Fe3 O4 /Ag, and (3) CV + Fe3 O4 /Ag + GSH (0.1 mmol L−1 ). Other experimental conditions: CV 2 mg L−1 ; substrate Fe3 O4 /Ag 125 mg L−1 ; CV pre-adsorption time 15 min, GSH adsorption time 20 min.
Fig. 5. Effects of CV concentration (a) and pre-adsorption time (b) on the SERS intensity of CV at 1620 cm−1 . Major experimental conditions: Fe3 O4 /Ag 25 mg L−1 , CV pre-adsorption time 15 min (a), and CV 1 mg L−1 in (b). The time dependence of the absorbance (at 591 nm) of 2 mg L−1 CV of the residual solution was also given in (b) after the substrate particles (load 125 mg L−1 ) were removed off. Fig. 3. A comparison between SERS spectra of (1) Fe3 O4 /Ag and (2) GSH (0.5 mol L−1 ), and (3) Raman spectrum of solid GSH.
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Fig. 6. Time dependence of SERS intensity of adsorbed CV and the absorbance of CV in residual solution. Fe3 O4 /Ag 25 mg L−1 , CV 1 mg L−1 , GSH 1 mol L−1 , CV preadsorption time 15 min.
increasing CV concentration up to 1 mg L−1 , and then became saturated beyond this concentration. Therefore, the concentration of the CV probe was optimized at 1 mg L−1 (2.5 mol L−1 ) in the present work. Similarly, the effect of pre-adsorption time was checked. As shown in Fig. 5b, the SERS intensity of CV was rapidly decreased with prolonging the pre-adsorption time, and became saturation at 15 min, which corresponded to the time being required for the adsorption/desorption equilibrium of CV on the surface of the Fe3 O4 /Ag substrate. At the same time, we also monitored the absorbance of the residual solution (at 591 nm) after the solid particles were removed (Fig. 5b). This also confirmed that the adsorption/desorption equilibrium of CV on the surface of the substrate was achieved at 15 min. Therefore, the pre-adsorption time was optimized at 15 min. 3.4. Effect of GSH adsorption time The time profiles of the SERS intensity of adsorbed CV and the absorbance of CV in residual solution after GSH added were recorded in Fig. 6. It was observed that the SERS intensity of CV was decreased and the absorbance of CV in residual solution was increased with increasing the GSH adsorption time up to 20 min. Beyond 20 min, both of the two curves in Fig. 6 went to a platform. The appearing of the platform signed the ending of the replacement of the adsorbed of CV by GSH. Therefore, the GSH adsorption time was optimized at 20 min.
Fig. 7. (a) SERS spectra of CV in the presence of GSH recorded under the optimal conditions (Fe3 O4 /Ag 25 mg L−1 ; CV 1 mg L−1 ; pre-adsorption time 15 min; GSH adsorption time 20 min). (b) A plot of 1620 cm−1 Raman intensity decrease (I0 –I) against the concentration of GSH under the optimal conditions. The inset in (b) gave a linear correlation between the intensity decrease of the CV SERS signal (I0 –I) and the concentration of GSH in the range of 50–700 nmol L−1 .
coefficient R2 = 0.992). The detection limit of GSH was estimated to be 40 nmol L−1 according to signal/noise = 3. Since the operation of the proposed method was simple and fast with a good sensitivity, this strategy shows a good prospect in practical application. 3.6. Selectivity of the proposed method
3.5. SERS detection of GSH Under the optimized conditions (Fe3 O4 /Ag 25 mg L−1 ; CV concentration 1 mg L−1 ; pre-adsorption time 15 min; GSH adsorption time 20 min), SERS spectra of CV were recorded in the presence of GSH at various concentrations as illustrated in Fig. 7a. By selecting the peak at 1620 cm−1 , the decrease in the peak intensity (I0 –I) was plotted against the concentration of GSH in Fig. 7b. It was easily observed that the SERS signal was decreased rapidly as the concentration of GSH was increased up to about 10 mol L−1 . When the concentration of GSH was increased to 20 mol L−1 , the SERS intensity almost faded off completely, indicating that almost all the pre-adsorbed CV molecules on the substrate had been replaced by GSH molecules. The inset in Fig. 7b gave a linear correlation between the decrease degree of the CV SERS signal and the concentration of GSH in the range of 50–700 nmol L−1 (the regression
The selectivity for GSH of the proposed SERS method was confirmed by measuring the responses of GSH in the presence of other common constituents in real biological samples. The influences of several inorganic anions and cations (NO3 − , Cl− , H2 PO4 − , HPO4 2− , HCO3 − , K+ , Na+ , Ca2+ , Zn2+ ) at a concentration of 300 mol L−1 were measured on the detection of 0.3 mol L−1 GSH. As shown in Fig. 8a, the interferences of these inorganic ions were negligible. According to the mechanism of the proposed methods, small organic molecules containing N or/and S elements like amino acids may disturb the detection. However, we observed that the co-existence of individual S-free amino acids (glycline, serine, threonine, phenylalanine, tyrosine, and tryptophan) at a concentration of 300 nmol L−1 produced little interference on the analysis results of 300 nmol L−1 GSH, because the induced error was less than 5% (see Fig. 8b). Even for the S-containing molecules (methionine, cystine, GSSG), especially the molecules contains free–SH group such
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Fig. 8. Effects of (a) inorganic ions (300 mmol L−1 ) and (b) bioorganic molecules (300 nmol L−1 ) on Raman intensity decrease of 1 mg L−1 CV in presence of 300 nmol L−1 GSH.
as cysteine and N-acetylcysteine, their addition (300 nmol L−1 ) only produced a relative error of around +10% to the intensity decrease in the SERS signal induced by 300 nmol L−1 GSH. Such preferential replacement by GSH as compared to cysteine or N-acetylcysteine is due to its special molecule structure with a higher binding affinity toward Ag through multiple functional groups, including the thiol groups [43]. This high affinity allows selective detection of GSH in the presence of interfering thiol-based biomolecules. Because the concentration of the related small molecules cysteine and Nacetylcysteine in bio-tissues and serum is below the concentration of GSH, their interference would be negligible in the practical analysis. 3.7. Stability and reproducibility of the method SERS is highly sensitive to the chemical environment, the non-uniformity of the substrate often leads to poor stability and reproducibility, which decreases the credibility for quantitative detection. In our proposed method, Fe3 O4 nanoparticles synthesized with the assistance of ultrasound had a uniform size distribution, this guaranteed the uniform deposition of Ag on Fe3 O4 nanoparticles. To examine the stability we obtained the substrate with a mapping scanning with an area of 50 m × 50 m. As shown in Fig. 9a, the SERS intensity (at 1620 cm−1 ) of CV (1 mg L−1 ) in the presence of 500 nmol L−1 GSH almost did not changed in the scanned area with a relative standard deviation (RSD) of about 10.1%. Then, we synthesized 5 batches of Fe3 O4 /Ag and monitored
Fig. 9. (a) SERS intensity of 1 mg L−1 CV at 1620 cm−1 with addition of 500 nmol L−1 GSH by map scanning. Step length 5 m, Fe3 O4 /Ag 25 mg L−1 , CV pre-adsorption time 15 min, GSH adsorption time 20 min; (b) Raman intensity of 1 mg L−1 CV at 1620 cm−1 measured from 5 batches of substrate with addition of GSH. Fe3 O4 /Ag 25 mg L−1 , cGSH 0.2–20 mol·L−1 , CV pre-adsorption time 15 min, GSH adsorption time 20 min.
the displacement process by adding GSH. As shown in Fig. 9b, the time profiles of the SERS signal 1620 cm−1 almost completely coincided between these batches, with a RSD value of 7.1%. The above results confirmed that the proposed method had a good reproducibility. Table 1 compared the new method with some methods reported on literature. It clearly showed that the SERS method was very sensitive and our SERS method was superior to other reported SERS method in reproducibility. 3.8. Determination of GSH and GSSG in real biological samples The concentration and molar ratio of GSH to GSSG are important indicators of health. The previously reported methods for the determination of GSH and GSSG in bio-matrix often require complex operation procedures such as thiol blocking and derivatization. However, the coexistence of GSSG and other thiols does not interfere with the detection of GSH in our proposed method, as shown in Fig. 8b. After the concentration of GSH is quantified, the GSSG is reduced to GSH by borohydride reduction, and then the newly generated GSH is further quantified, which gives the concentration of GSSG. Using the proposed SERS method, we determined the
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Table 1 A comparison of the proposed method with other methods for GSH detection. Method
Linear range (mol L−1 )
Detection limit (mol L−1 )
Reproducibility RSD/%
Reference
Enzymatic HPLC CE MS Photoelectrochemical LC FL Electrochemical SERS (direct) SERS (dye probe) SERS
200–1000 0.07–10 1.58–200 0.1–100 0.01–10 24–214 0.06–10 0.3–5000 – 5–200 0.05–0.7
– 0.07 0.62 0.03 0.01 8.3 0.02 2.2 500 1 0.04
1.9 (n = 5) 1.4 (n = 4) – 10.2 (n = 6) – – – – – – 7.1 (n = 10)
[5] [9] [44] [45] [46] [13] [6] [14] [25] [29] this work
Table 2 Concentrations of GSH and GSSG in human whole blood and hepatoma cell HepG2 and Be17402. Blood sample 1 −1
GSH (mol L ) GSSG (mol L−1 ) GSH (mol/cell) GSSG (mol/cell) GSH/GSSG a
494.0 ± 23.6 (587.0 ± 51.0) 72.0 ± 9.8
6.8
Blood sample 2 a)
438.4 ± 23.2 (426.0 ± 8.0) 76.8 ± 8.4
HepG2 cell
Be17402 cell
(5.0 ± 0.6) × 10−9 (3.2 ± 0.3) × 10−11 156.2
(3.4 ± 0.7) × 10−9 (2.3 ± 0.4) × 10−11 147.8
a)
5.7
The data in the parenthesis of GSH in the blood samples were obtained with the HPLC method.
concentrations of GSH and GSSG in the human whole blood samples and the extract of hepatoma cells HepG2 and Be17402. The results were shown in Table 2. As a control experiment, the determination of GSH in blood samples was also performed by using the HPLC and fluorometric detector following precolumn derivatization with o-phthalaldehyde [10]. Since the concentration of GSH in cells extract was below the detection limit of the HPLC method, thus we qualified the concentration in the extract of hepatoma cells only by SERS method. As shown in Table 2, the concentrations of GSH and GSSG were in the range of 400–500 and 50–100 mol L−1 in human whole blood samples, while 3–5 and 2–3 fmol/cell in hepatoma cells HepG2 and Be17402, respectively. Because the detector integrated with our HPLC equipment was limited, we could not quantify GSSG in our laboratory. Therefore, Table 2 gave the concentrations of only GSH but not GSSG in blood samples obtained by the HPLC method. The results for the quantification of GSH obtained by our SERS method are in agreement with that by the HPLC method. Moreover, we conducted another control experiment of standard addition. By adding 300 mol L−1 GSH in to the two blood samples, the recovery of GSH was 99.0% and 106.3% from the two blood samples, further demonstrating that the proposed SERS method is qualified for the detection in real complex samples.
4. Conclusions Fe3 O4 /Ag nanoparticles with a uniform size distribution were prepared as SERS substrate. The characterization indicated that metallic Ag was uniformly deposited on the Fe3 O4 magnetic nanoparticles. CV is Raman sensitive, and the Fe3 O4 /Ag nanoparticles produced much enhanced SERS responses of CV. In comparison with CV, GSH is not a Raman sensitive compound. However, the adsorption of GSH the Fe3 O4 /Ag nanoparticles though Ag-S bonding was much stronger than that of CV though Ag-N bonding. Therefore, the pre-adsorbed CV molecules on the surface of Fe3 O4 /Ag nanoparticles were replaced by the newly added GSH molecules. This resulted in a drastic decrease in the SERS intensity of CV. By combining the Fe3 O4 /Ag substrate and the CV Raman probe, the present work proposed a facile SERS method to detect GSH at low concentrations. After optimizing the experimental conditions, the new method could detect trace GSH at concentrations in the concentration range of 50–700 nmol L−1 with a detection limit of
40 nmol L−1 . The proposed method was applied to determine GSH and GSSG in the real samples of blood and cells, giving satisfactory analytical results and showing the merits of the method such as a good sensitivity, stability and reproducibility. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 Program) (Grant No. 2012AA06A304), and the National Science Foundation of China (Grant No. 21177044 and 21377169). References [1] T.M. Bray, C.G. Taylor, Can. J. Physiol. Pharmacol. 71 (1993) 746–751. ˜ [2] M. Asensi, J. Sastre, F.V. Pallardo, A. Lloret, M. Lehner, J.G. Asuncion, J. Vina, Methods Enzymol. 299 (1999) 267–276. [3] S.Y. Zhu, X.E. Zhao, W. Zhang, Z.Y. Liu, W.J. Qi, S. Anjum, G.B. Xu, Anal. Chim. Acta 786 (2013) 111–115. [4] T. Yamaguchi, I. Katoh, S. Kurata, Eur. J. Biochem. 269 (2002) 2782–2788. [5] S. Timur, D. Odaci, A. Dincer, F. Zihnioglu, A. Telefoncu, Talanta 74 (2008) 1492–1497. [6] T.H. Chen, W.L. Tseng, Small 8 (2012) 1912–1919. [7] X.Q. Chen, Y. Zhou, X.J. Peng, J.Y. Yoon, Chem. Soc. Rev. 39 (2010) 2120–2135. [8] C. Xie, D.F. Zhong, X.Y. Chen, Anal. Chim. Acta 788 (2013) 89–98. [9] G.P. McDermott, J.M. Terry, X.A. Conlan, N.W. Barnett, P.S. Francis, Anal. Chem. 83 (2011) 6034–6039. [10] D. Tsikas, J. Sandmann, D. Holzberg, P. Pantazis, M. Raida, J.C. Frolich, Anal. Biochem. 273 (1999) 32–40. [11] Y. Wang, Y. Xie, M. Bernier, I.W. Wainer, J. Chromatogr. A 1216 (2009) 3533–3537. [12] D.B. Liu, W.W. Chen, Y. Tian, S. He, W.F. Zheng, J.S. Sun, Z. Wang, X.Y. Jiang, Adv. Healthcare Mater. 1 (2012) 90–95. [13] L.L. Liu, Q. Ma, Y. Li, Z.P. Liu, X.G. Su, Talanta 114 (2013) 243–247. [14] P.T. Lee, R.G. Compton, Electroanalysis 25 (2013) 1613–1620. [15] Y.Q. Hao, D.D. Xiong, L.Q. Wang, W.S. Chen, B.B. Zhou, Y.N. Liu, Talanta 115 (2013) 253–257. [16] Y. Wang, J. Lu, L.H. Tang, H.X. Chang, J.H. Li, Anal. Chem. 81 (2009) 9710–9715. [17] B.K. McMahon, T. Gunnlaugsson, J. Am. Chem. Soc. 134 (2012) 10725–10728. [18] B.Y. Han, J.P. Yuan, E.K. Wang, Anal. Chem. 81 (2009) 5569–5573. [19] H. Xu, M. Hepel, Anal. Chem. 83 (2011) 813–819. [20] F. Tanaka, N. Mase, C.F. Barbas III, Chem. Commun. 15 (2004) 1762–1763. [21] M.K. Fan, G.F.S. Andrade, A.G. Brolo, Anal. Chim. Acta 693 (2011) 7–25. [22] N.P.W. Pieczonka, R.F. Aroca, Chem. Soc. Rev. 37 (2008) 946–954. [23] S.F. Zong, Z.Y. Wang, H. Chen, J. Yang, Y.P. Cui, Anal. Chem. 85 (2013) 2223–2230. [24] M.L. Cheng, B.C. Tsai, J. Yang, Anal. Chim. Acta 708 (2011) 89–96. [25] M. Larsson, J. Lindgren, J. Raman Spectrosc. 36 (2005) 394–399. [26] G.G. Huang, X.X. Han, M.K. Hossain, Y. Ozaki, Anal. Chem. 81 (2009) 5881–5888. [27] Y.C. Cao, R.C. Jin, J.M. Nam, C.S. Thaxton, C.A. Mirkin, J. Am. Chem. Soc. 125 (2003) 14676–14677.
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A surface-enhanced Raman scattering method for detection of trace glutathione on the basis of immobilized silver nanoparticles and crystal violet probe Lei Ouyang a,b , Lihua Zhu a,b , Jizhou Jiang a , Heqing Tang b,∗ a
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P.R. China Key Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission and Ministry of Education, College of Chemistry and Materials Science, South Central University for Nationalities, Wuhan 430074, P.R. China b
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
• A SERS substrate was synthesized by fixing Ag onto Fe3 O4 magnetic particles. • The analysis of Raman insensitive GSH was achieved with Raman sensitive crystal violet as a probe. • The new method can determine GSH in the range of 50–700 nmol L−1 with a detection limit of 40 nmol L−1 . • The proposed method has good selectivity and reproducibility for trace GSH detection in real samples.
a r t i c l e
i n f o
Article history: Received 18 September 2013 Received in revised form 19 January 2014 Accepted 24 January 2014 Available online 3 February 2014 Keywords: Surface-enhanced Raman scattering Glutathione Immobilized silver nanoparticles Crystal violet Replacement.
a b s t r a c t Unsatisfactory sensitivity and stability for molecules with low polarizability is still a problem limiting the practical applications of surface-enhanced Raman scattering (SERS) technique. By preparing immobilized silver nanoparticles (Fe3 O4 /Ag) through depositing silver on the surface of magnetite particles, a highly sensitive and selective SERS method for the detection of trace glutathione (GSH) was proposed on the basis of a system of Fe3 O4 /Ag nanoparticles and crystal violet (CV), in which the target GSH competed with the CV probe for the adsorption on the Fe3 O4 /Ag nanoparticles. Raman insensitive GSH replaced the highly Raman sensitive CV adsorbed on the surface of Fe3 O4 /Ag particles. This replacement led to a strong decrease of the CV SERS signal, which was used to determine the concentration of GSH. Under optimal conditions, a linear response was established between the intensity decrease of the CV SERS signal and the GSH concentration in the range of 50–700 nmol L−1 with a detection limit of 40 nmol L−1 . The use of a Fe3 O4 /Ag substrate provided not only a great SERS enhancement but also a good stability, which guarantees the reproducibility of the proposed method. Its use for the determination of GSH in practical blood samples and cell extract yielded satisfactory results. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +86 27 67843323; fax: +86 27 67843323. E-mail addresses: [email protected], [email protected] (H. Tang). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.046
Glutathione (␥-L-glutamyl-L-cysteinyl-glycine; GSH) is the most abundant nonprotein thiol source in mammalian tissues and plays a pivotal role in biological processes [1]. It has both reduced form (GSH) and oxidized dimeric form (GSSG). By the conversion of the two forms, it can maintain thiol-disulfide reduction–oxidation potential. It also performs as an important
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intracellular reduction–oxidation buffer, which may trap free radicals and peroxides to prevent cell damage [2]. The ratio of GSH/GSSG reflects the ability of antioxidant and is able to be used for monitoring the cells overall health. GSH has many other biological functions like detoxication, signal transduction, and transportation/elimination of metal ions since thiol is involved in most of biological processes [3]. Medical studies show that the level of GSH in plasma is related to diseases such as HIV, Alzheimer’s disease, and Parkinson’s disease [4]. Hence it is needed to develop sensitive and convenient methods for the detection of GSH in biological matrix. The detection of GSH and other thiols can be realized by using enzymatic [5], florescence (FL) [6,7], mass spectrometric (MS) methods [8]. The separation methods using liquid chromatography (LC) and capillary eletrophoresis (CE) are most popular for selective and sensitive determinations of GSH, giving the methods such as LC-chemiluminiscence (LC-CL) [9], LC-florescence (LC-FL) [10], CE-laser induced FL [11]. Although a number of techniques have been employed to assay the thiols, some problems still occur due to the operation of sophisticated instrumentation and/or the complexity of the procedure. Another strategy for the detection of GSH without separation is feasible by taking account of its reactive sulfydryl (–SH) group. This group has a strong interaction with many metal ions such as Hg, Au and Ag [12] and many organofunctional groups such as aldehyde group. These interactions are used to develop CL [13], electrochemical [14] and colorimetric [15] methods for quantifying GSH. Wang et al. synthesized grapheneCdTe composite with a selective combination to –SH, and used it as an electrochemical probe for GSH detection [16]. By using a fluorescence probe containing Tb(III), McMahon et al. determined the concentration of GSH [17]. Han et al. prepared Hg-modified CdTe quantum dot with a high sensitivity to biological thiol [18]. Taking account of the different association constant of DNA and GSH with Hg ions, Xu et al. detected GSH by using the replacement reaction between DNA and GSH [19]. Since the environmental risk of heavy metals, some heavy-metal-free probes have also been proposed. Tanaka et al. used an aldehyde to react with GSH, and used the induced fluorescence decrease to detect the concentration of GSH [20]. However, these probes for GSH usually consisted of short wavelength emitting and short lifetime-based fluorophores, which showed many drawbacks when used in biologic media. Surface-enhanced Raman scattering (SERS) is a sensitive analytical technique [21,22], and it is especially advantageous for treating biological samples because it can analyze aqueous samples without disturbance of water [23,24]. SERS should have a prospect in GSH detection. However, due to the low polarization of GSH, its direct SERS detection is not sensitive. Larsson et al. reported that the SERS detection of GSH on nano silver had a limit of detection as high as 0.5 mmol L−1 [25]. A heat-induced SERS method was reported to detect low level GSH, but suffering strong interferences [26]. A Raman-probe mediated method is helpful to overcome the difficulty from low polarizing molecules [27,28]. On a nano silver substrate, Huang et al. used rhodamine B as probe for the detection of GSH and made a great achievement by decreasing the detect limit to 1 mol L−1 GSH [29]. However, according to our experiences, simple nano silver gel is not a good SERS substrate for liquid detection, because the nano particles exhibit low uniformity and low stability, being unfavorable to the reproducibility of the analytical method [30]. Therefore, it is needed to develop better SERS substrate. Silver- and gold-coated iron oxides were recently reported as SERS substrate due to their wide range of potential uses in industrial, biomedical, and environmental applications [31,32]. Du et al. prepared Fe3 O4 /Ag as a substrate for the detection of PAHs and Cr(VI) [33,34]. Zhou et al. synthesized core-shell Fe3 O4 /Au for the protein detection [35]. Shen et al. used Fe3 O4 /Ag/SiO2 /Au
as substrate for the study of long-range plasmon coupling effect on the SERS responses [36]. These nanocomposites combined the advantages of “core” and “shell”, providing such composites with enhanced stabilization as well as SERS effect and greatly extending the application of such substrate. When nanocomposites are used as a SERS substrate, their uniform particle size distribution is very important for the reproducibility of the SERS method. In the present work, we tried to develop a better substrate and find a better Raman probe for sensitive and indirect detection of GSH. In consideration of the reported methods of nano gold/silver fixing and our experiences in the ultrasonic preparation of Fe3 O4 particles [37], we prepared Fe3 O4 particles with a narrow particle size distribution, and then synthesized Fe3 O4 /Ag as a SERS substrate by in-situ reduction of silver ions. Fe3 O4 /Ag particles are easily magnetized by an external magnetic field, being favorable to the localization when they serve as an efficient SERS substrate. We chose Raman sensitive molecule crystal violet (CV) as a Raman probe. The purple dye CV can easily adsorb to the noble metal substrate and has a maximum absorption wavelength around 591 nm, which matches with the laser wavelength of 532 nm being used for the SERS measurement. These properties of CV make it a good Raman probe in SERS [38,39]. By combining these two selections (the Fe3 O4 /Ag substrate and CV probe), a sensitive SERS method was developed for analyzing GSH with good reproducibility and anti-interference ability. 2. Experimental 2.1. Reagents Ferric chloride (FeCl3 ), ferrous sulfate (FeSO4 ), silver nitrate (AgNO3 , >99.8%), sodium hydroxide (NaBH4 , >99.5%), aqueous ammonia (25%), sodium chloride, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium hydrogen carbonate, sodium nitrate, sodium chloride, calcium nitrate tetrahydrate, zinc nitrate hexahydrate, potassium nitrate, trichloroacetic acid (TCA), CV and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd (China). 3Aminopropyltrimethoxysilane (APTMS, 97%), cystine (Cyss), GSSG, N-acetylcysteine (NAC) was from Aladdin Industrial Corporation (USA). GSH was from Bio Basic Inc. (Canada). Cysteine (Cys), glycline (Gly), serine (Ser), threonine (Thr), glutamine (Glu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) were obtained from Shanghai Ruji Biological Technology Development Co., Ltd. (China). All reagents were of analytical reagent grade and used without further purification. 2.2. Preparation of Fe3 O4 MNPs Fe3 O4 nanoparticles were synthesized by an ultrasonic assisted reverse co-precipitation method [37]. In brief, 5.0 mL of 1.0 mol L−1 FeCl3 solution and 10.0 mL of 0.5 mol L−1 FeSO4 solutions were first mixed and then added dropwise into 20 mL of 3.5 mol L−1 ammonia water at 60 ◦ C with ultrasound irradiation for 30 min which was carried out in an ultrasound clean bath operating at 25 kHz with a power of 140 W (KQ-200KDE, China). The produced black Fe3 O4 nanoparticles were collected by magnetic separation, washed with water to neutral pH, and dried in a vacuum oven at 60 ◦ C. 2.3. Preparation of Fe3 O4 /Ag particles Fe3 O4 (0.05 g) was dispersed in 20 mL ethanol and sonicated for 5 min. After the addition of 200 L APTMS, the solution was vigorously mixed for 6 h. The APTMS modified Fe3 O4 particles were separated by an applied magnetic field and washed with ethanol and water. The next procedure was in-situ reduction Ag+ to Ag. The
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APTMS-coated Fe3 O4 (0.05 g) was dispersed in 20 mL of 0.1 mol L−1 AgNO3 , and the reaction was initiated by adding 20 mL 0.2 mol L−1 NaBH4 dropwise into the solution with stirring, being followed by further stirring for 45 min. The particles were then obtained with an applied magnetic field and washed fully with water. Finally, the obtained Fe3 O4 /Ag NPs were re-dispersed in 50 mL of water.
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3. Result and discussion 3.1. Substrate characterization
A GSH stock solution of 0.01 mol L−1 was prepared by dissolving GSH into diluted water. The Fe3 O4 /Ag dispersion was pre-equilibrated with an aqueous solution of the probe CV by mixing them before the detection of GSH and stirring for 15 min. Then the GSH sample solution was added to the CV solution, solution pH was adjusted to 6.5 with NaOH solution and the co-adsorption was conducted for another 20 min. The NPs were assembled by using an applied magnetic field for SERS detection. Part of the particles with solution (10 L) was taken out for SERS detection. All SERS and conventional Raman spectra were measured with a DXR Raman Microscopy equipped with a CCD detector (Thermo Fisher Scientific, USA). The 532 nm line of NIR laser was used as an excitation source. The laser power was 3.0 mW at the sample position and the exposure time for each SERS measurement was set at 2 s with 2 accumulations in this study.
As shown in Fig. 1a, there were a series of characteristic peaks at 2 = 30.4◦ , 35.7◦ , 43.5◦ , 53.9◦ , 57.15◦ and 63.1◦ in the patterns of both Fe3 O4 and Fe3 O4 /Ag, which fit well with XRD PDF card 750449 of Fe3 O4 . Unlike Fe3 O4 , the Fe3 O4 /Ag sample also gave a series of peaks at 2 = 38.2◦ , 44.4◦ , 64.6◦ and 77.6◦ , which are the characteristic peaks of Ag according to XRD PDF card 87-0720. This signs the deposition of Ag on the surface of Fe3 O4 MNPs. The average core size of the particles was evaluated with the Debye–Sherrer formula D = K/(ˇ cos), where K is the Sherrer constant (0.89), is the X-ray wavelength (0.15418 nm), ˇ is the peak full width at half maximum (FWHM), and is the Bragg diffraction angle. From the peak (3 3 1) of Fe3 O4 and (1 1 1) of Ag, the average crystal size of Fe3 O4 and Fe3 O4 /Ag were calculated to be 16.22 and 24.18 nm, respectively. XPS was performed to provide elemental information of the surface atomic composition of Fe3 O4 /Ag. Fig. 1b exhibited the XPS spectrum of Fe3 O4 /Ag, which clearly showed the signals for Fe, Ag, O, and C element, the atomic percentage of each element was 25.82%, 5.12%, 50.29% and 18.77%. The XPS spectrum of Fe (2p) and Ag (3d) were shown in Fig. 1c and d. The Fe 2p1/2 and 2p3/2 peaks were centered at 724.2 eV and 710.8 eV, respectively, which were characteristics of Fe3 O4 [37]. This suggests that no significant change in peak position was observed upon the coating of Ag. The Fe (2p) spectra were deconvoluted by taking into account contributions from Fe(II), Fe(III) and the satellite peak. The Fe (2p) peaks at binding energies of 711.2 eV and 725.4 eV with a satellite signal at 718.2 eV were characteristic of Fe(III), while the peaks of 709.9 eV and 723.4 eV with a satellite signal at 713.4 eV were characteristic of Fe(II). The atomic ratio of Fe(III) to Fe(II) was about 2:1, confirming the formation of Fe3 O4 . In the envelop of Ag (3d), two peaks located at 367.6 and 373.6 eV with a spin-orbit splitting of 6 eV, showing the formation of Ag layer [40]. Both the XRD and XPS results demonstrate that metallic of Ag was coated on the Fe3 O4 surface. On a laser scattering particle size distribution analyzer, the average sizes of Fe3 O4 and Fe3 O4 /Ag particles were measured to be 345 nm and 260 nm, respectively. This hints that the deposition of Ag is favorable to depressing the aggregation of Fe3 O4 particles, possibly due to the capping effect of the introduced APTMS for the deposition of Ag.
2.6. Extract collection and analysis of whole blood samples and hepatoma cells
3.2. Displacement effect of GSH on Raman responses of CV and the possible mechanism
The method of McDermott et al. [9] was followed for collecting and processing blood samples. The blood samples (3 mL) were drawn from several healthy adult volunteers and put into the blood tubes immediately, then reserved at 0–4 ◦ C. Noticing that the perchloric acid may oxidize GSH during the adsorption process, we used TCA instead of perchloric acid, which was used in the method of McDermott et al. [9]. For whole blood analysis, 500 L of blood was added into the eppendorf tube with 500 L TCA (10%, w/v), the tubes were then vortexed and incubated on ice for 10 min. After the blood proteins were precipitated, the suspensions were then centrifuged at 14,000 rpm for 5 min, and the supernatant was diluted and analysed with the proposed method. Hepatoma cell HepG2 and Be17402 were collected after trypsinization and washed with phosphate buffer (PBS) (pH 7.4). After counting, the cells were centrifuged. The precipitated pellet was mixed with 10% TCA and reacted for 6 h with stirring. Then the cells were centrifuged, the supernatant was collected. The pellet was washed with TCA and the supernatant was mixed with the former supernatant for GSH detection.
CV is a common cationic dye with a symmetry molecule structure. The solution of CV at a concentration of 1 mg L−1 (2.4 mol L−1 ) yielded very weak Raman response (curve 1 in Fig. 2a). By using the Fe3 O4 /Ag substrate (load 25 mg L−1 ), the Raman signal of CV was enhanced by a factor of about 105 (curve 2 in Fig. 2a), demonstrating that the Fe3 O4 /Ag particles are an excellent SERS substrate and CV is a good Raman probe. After the addition 10 mol L−1 of GSH, the SERS intensity of CV on the Fe3 O4 /Ag substrate was decreased significantly by a factor of 10 (curve 3 in Fig. 2a). The significant displacement effect of GSH on the SERS responses of CV was attributed to the fact that less Raman active GSH adsorbs more strongly on the surface of the Fe3 O4 /Ag substrate than CV does, as discussed below. The adsorption competition between GSH and CV was confirmed by comparing the related UV-vis absorption spectra. As shown in Fig. 2b, CV (2 mg L−1 ) had a strong absorption band with the maximum absorption wavelength at 591 nm (curve 1). After CV (2 mg L−1 ) was mixed with Fe3 O4 /Ag (load 125 mg L−1 ) for 15 min, the UV-vis absorption spectrum of the residual solution
2.4. Characterization X-ray diffraction (XRD) patterns were recorded on an X’Pert PRO X-ray diffractometer (PANalytica) with a Cu K␣ radiation source generated at 40 kV and 30 mA. The particle size distribution was measured by a dynamic light-scattering method on a ZEN 3690 Zetasizer (Malvern, United Kingdom). The surface chemical compositions of Fe3 O4 /Ag MNPs were identified by X-ray photoelectron spectroscopy (XPS, XSAM 800, KROTOS) using the Mg K␣ line (1253.6 eV) as an excitation source. The pressure in the XPS analysis chamber was maintained at 10−9 mbar. The binding energies of all peaks were referenced to the C (1s) line (285.0 eV). Before the XRD and XPS measurements, the prepared Fe3 O4 /Ag nanoparticles were collected by magnetic separation and vacuum dried at 50 ◦ C for 1 h. Such a pre-treatment could not cause the change of the crystalline structure and the oxidation of metallic silver in the Fe3 O4 /Ag samples. 2.5. SERS analysis
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Fig. 1. (a) XRD patterns of Fe3 O4 and Fe3 O4 /Ag. (b) XPS survey scan spectrum, (c) Fe 2p envelop and (d) Ag 3d envelop of Fe3 O4 /Ag.
was recorded after the solid particles were removed off (curve 2). In comparison with the CV solution, the absorbance of the residual solution was decreased, indicating that the CV concentration in the residual solution was decreased due to its adsorption on the substrate. In the third run, 0.1 mmol L−1 of GSH was mixed with the mixture of CV and Fe3 O4 /Ag, and then the UV-vis absorption spectrum of the second residual solution was recorded (curve 3) after the solid particles were removed off. By comparing curves 2 and 3 in Fig. 2b, it was found that the CV concentration was recovered to an extent in the second residual solution. This hinted that parts of the adsorbed CV molecules were released from the substrate surface due to the stronger adsorption of GSH. To make clear the mechanism of the replacement, we made a comparison between SERS spectra of Fe3 O4 /Ag and GSH (0.5 mol L−1 ) with the Raman spectrum of solid GSH. As shown in Fig. 3, in the Raman spectrum of solid GSH, there was a characteristic peak at 2571 cm−1 , corresponding to the stretch vibration of–SH. This peak disappeared in the SERS spectrum of and GSH on Fe3 O4 /Ag, indicating that the –SH group was eliminated by the adsorption of GSH on the surface of the substrate. At the same time, a new peak appeared at 236 cm−1 in the SERS spectrum of GSH, which corresponded to the Ag-S bond. This supported that the adsorption of GSH on the Fe3 O4 /Ag was achieved by forming Ag-S bond. The mechanism of Ag-S bond formation was further clarified by using SCN− (25 mmol L−1 ) as a probe. Since the stretch vibration of
C N (CN ) is very sensitive to the coordination environment, the electron transformation of targeting molecule could be studied by observing the variations in CN [41]. As shown in Fig. 4, the characteristic peak of CN appeared at 2117 cm−1 , but the addition of GSH made the peak red shift. When the GSH concentration was increased to 10−3 mol L−1 , the red shift was increased to 20 cm−1 . This phenomenon could be explained in terms of the electrondonor effect of GSH: The S atom of the adsorbed GSH donated its lone pair electrons to silver surface, and then the negative charge on the silver surface transferred to SCN− through the generated S-Ag bond. The conjugated effect between S atom and C N bond made it possible to increase the electron density of C N bond and consequently weaken the C N bond and decreased the frequency of C N. This GSH-induced red shift of CN confirmed again that the GSH interacted with Fe3 O4 /Ag by forming Ag-S through the donation effect of the S atom in the GSH molecule. Since the Ag-N bond is weaker than the Ag-S bond [42], the replacement of Ag-N by Ag-S could take place, which led to the decrease of SERS signals of CV. This displacement effect could be used to detect GSH indirectly. 3.3. Effect of CV concentration and adsorption time To optimize the concentration of the probe CV, the concentration dependence of its SERS intensity at 1620 cm−1 was investigated with a load of 25 mg L−1 Fe3 O4 /Ag as the substrate. As shown in Fig. 5a, the SERS intensity was increased with
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Fig. 4. SERS spectra of SCN− (0.025 mol L−1 ) in presence different concentrations of GSH.
Fig. 2. (a) A comparison between (1) Raman spectrum of CV, (2) SERS spectrum of CV and (3) SERS spectrum of CV in the presence of 10 mol L−1 GSH. Other experimental conditions: substrate Fe3 O4 /Ag 25 mg L−1 ; CV concentration 1 mg L−1 ; CV pre-adsorption time 15 min; GSH adsorption time 20 min. (b) UV-vis absorption spectra of the residual CV in solution (particles have been removed): (1) CV, (2) CV + Fe3 O4 /Ag, and (3) CV + Fe3 O4 /Ag + GSH (0.1 mmol L−1 ). Other experimental conditions: CV 2 mg L−1 ; substrate Fe3 O4 /Ag 125 mg L−1 ; CV pre-adsorption time 15 min, GSH adsorption time 20 min.
Fig. 5. Effects of CV concentration (a) and pre-adsorption time (b) on the SERS intensity of CV at 1620 cm−1 . Major experimental conditions: Fe3 O4 /Ag 25 mg L−1 , CV pre-adsorption time 15 min (a), and CV 1 mg L−1 in (b). The time dependence of the absorbance (at 591 nm) of 2 mg L−1 CV of the residual solution was also given in (b) after the substrate particles (load 125 mg L−1 ) were removed off. Fig. 3. A comparison between SERS spectra of (1) Fe3 O4 /Ag and (2) GSH (0.5 mol L−1 ), and (3) Raman spectrum of solid GSH.
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Fig. 6. Time dependence of SERS intensity of adsorbed CV and the absorbance of CV in residual solution. Fe3 O4 /Ag 25 mg L−1 , CV 1 mg L−1 , GSH 1 mol L−1 , CV preadsorption time 15 min.
increasing CV concentration up to 1 mg L−1 , and then became saturated beyond this concentration. Therefore, the concentration of the CV probe was optimized at 1 mg L−1 (2.5 mol L−1 ) in the present work. Similarly, the effect of pre-adsorption time was checked. As shown in Fig. 5b, the SERS intensity of CV was rapidly decreased with prolonging the pre-adsorption time, and became saturation at 15 min, which corresponded to the time being required for the adsorption/desorption equilibrium of CV on the surface of the Fe3 O4 /Ag substrate. At the same time, we also monitored the absorbance of the residual solution (at 591 nm) after the solid particles were removed (Fig. 5b). This also confirmed that the adsorption/desorption equilibrium of CV on the surface of the substrate was achieved at 15 min. Therefore, the pre-adsorption time was optimized at 15 min. 3.4. Effect of GSH adsorption time The time profiles of the SERS intensity of adsorbed CV and the absorbance of CV in residual solution after GSH added were recorded in Fig. 6. It was observed that the SERS intensity of CV was decreased and the absorbance of CV in residual solution was increased with increasing the GSH adsorption time up to 20 min. Beyond 20 min, both of the two curves in Fig. 6 went to a platform. The appearing of the platform signed the ending of the replacement of the adsorbed of CV by GSH. Therefore, the GSH adsorption time was optimized at 20 min.
Fig. 7. (a) SERS spectra of CV in the presence of GSH recorded under the optimal conditions (Fe3 O4 /Ag 25 mg L−1 ; CV 1 mg L−1 ; pre-adsorption time 15 min; GSH adsorption time 20 min). (b) A plot of 1620 cm−1 Raman intensity decrease (I0 –I) against the concentration of GSH under the optimal conditions. The inset in (b) gave a linear correlation between the intensity decrease of the CV SERS signal (I0 –I) and the concentration of GSH in the range of 50–700 nmol L−1 .
coefficient R2 = 0.992). The detection limit of GSH was estimated to be 40 nmol L−1 according to signal/noise = 3. Since the operation of the proposed method was simple and fast with a good sensitivity, this strategy shows a good prospect in practical application. 3.6. Selectivity of the proposed method
3.5. SERS detection of GSH Under the optimized conditions (Fe3 O4 /Ag 25 mg L−1 ; CV concentration 1 mg L−1 ; pre-adsorption time 15 min; GSH adsorption time 20 min), SERS spectra of CV were recorded in the presence of GSH at various concentrations as illustrated in Fig. 7a. By selecting the peak at 1620 cm−1 , the decrease in the peak intensity (I0 –I) was plotted against the concentration of GSH in Fig. 7b. It was easily observed that the SERS signal was decreased rapidly as the concentration of GSH was increased up to about 10 mol L−1 . When the concentration of GSH was increased to 20 mol L−1 , the SERS intensity almost faded off completely, indicating that almost all the pre-adsorbed CV molecules on the substrate had been replaced by GSH molecules. The inset in Fig. 7b gave a linear correlation between the decrease degree of the CV SERS signal and the concentration of GSH in the range of 50–700 nmol L−1 (the regression
The selectivity for GSH of the proposed SERS method was confirmed by measuring the responses of GSH in the presence of other common constituents in real biological samples. The influences of several inorganic anions and cations (NO3 − , Cl− , H2 PO4 − , HPO4 2− , HCO3 − , K+ , Na+ , Ca2+ , Zn2+ ) at a concentration of 300 mol L−1 were measured on the detection of 0.3 mol L−1 GSH. As shown in Fig. 8a, the interferences of these inorganic ions were negligible. According to the mechanism of the proposed methods, small organic molecules containing N or/and S elements like amino acids may disturb the detection. However, we observed that the co-existence of individual S-free amino acids (glycline, serine, threonine, phenylalanine, tyrosine, and tryptophan) at a concentration of 300 nmol L−1 produced little interference on the analysis results of 300 nmol L−1 GSH, because the induced error was less than 5% (see Fig. 8b). Even for the S-containing molecules (methionine, cystine, GSSG), especially the molecules contains free–SH group such
L. Ouyang et al. / Analytica Chimica Acta 816 (2014) 41–49
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Fig. 8. Effects of (a) inorganic ions (300 mmol L−1 ) and (b) bioorganic molecules (300 nmol L−1 ) on Raman intensity decrease of 1 mg L−1 CV in presence of 300 nmol L−1 GSH.
as cysteine and N-acetylcysteine, their addition (300 nmol L−1 ) only produced a relative error of around +10% to the intensity decrease in the SERS signal induced by 300 nmol L−1 GSH. Such preferential replacement by GSH as compared to cysteine or N-acetylcysteine is due to its special molecule structure with a higher binding affinity toward Ag through multiple functional groups, including the thiol groups [43]. This high affinity allows selective detection of GSH in the presence of interfering thiol-based biomolecules. Because the concentration of the related small molecules cysteine and Nacetylcysteine in bio-tissues and serum is below the concentration of GSH, their interference would be negligible in the practical analysis. 3.7. Stability and reproducibility of the method SERS is highly sensitive to the chemical environment, the non-uniformity of the substrate often leads to poor stability and reproducibility, which decreases the credibility for quantitative detection. In our proposed method, Fe3 O4 nanoparticles synthesized with the assistance of ultrasound had a uniform size distribution, this guaranteed the uniform deposition of Ag on Fe3 O4 nanoparticles. To examine the stability we obtained the substrate with a mapping scanning with an area of 50 m × 50 m. As shown in Fig. 9a, the SERS intensity (at 1620 cm−1 ) of CV (1 mg L−1 ) in the presence of 500 nmol L−1 GSH almost did not changed in the scanned area with a relative standard deviation (RSD) of about 10.1%. Then, we synthesized 5 batches of Fe3 O4 /Ag and monitored
Fig. 9. (a) SERS intensity of 1 mg L−1 CV at 1620 cm−1 with addition of 500 nmol L−1 GSH by map scanning. Step length 5 m, Fe3 O4 /Ag 25 mg L−1 , CV pre-adsorption time 15 min, GSH adsorption time 20 min; (b) Raman intensity of 1 mg L−1 CV at 1620 cm−1 measured from 5 batches of substrate with addition of GSH. Fe3 O4 /Ag 25 mg L−1 , cGSH 0.2–20 mol·L−1 , CV pre-adsorption time 15 min, GSH adsorption time 20 min.
the displacement process by adding GSH. As shown in Fig. 9b, the time profiles of the SERS signal 1620 cm−1 almost completely coincided between these batches, with a RSD value of 7.1%. The above results confirmed that the proposed method had a good reproducibility. Table 1 compared the new method with some methods reported on literature. It clearly showed that the SERS method was very sensitive and our SERS method was superior to other reported SERS method in reproducibility. 3.8. Determination of GSH and GSSG in real biological samples The concentration and molar ratio of GSH to GSSG are important indicators of health. The previously reported methods for the determination of GSH and GSSG in bio-matrix often require complex operation procedures such as thiol blocking and derivatization. However, the coexistence of GSSG and other thiols does not interfere with the detection of GSH in our proposed method, as shown in Fig. 8b. After the concentration of GSH is quantified, the GSSG is reduced to GSH by borohydride reduction, and then the newly generated GSH is further quantified, which gives the concentration of GSSG. Using the proposed SERS method, we determined the
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Table 1 A comparison of the proposed method with other methods for GSH detection. Method
Linear range (mol L−1 )
Detection limit (mol L−1 )
Reproducibility RSD/%
Reference
Enzymatic HPLC CE MS Photoelectrochemical LC FL Electrochemical SERS (direct) SERS (dye probe) SERS
200–1000 0.07–10 1.58–200 0.1–100 0.01–10 24–214 0.06–10 0.3–5000 – 5–200 0.05–0.7
– 0.07 0.62 0.03 0.01 8.3 0.02 2.2 500 1 0.04
1.9 (n = 5) 1.4 (n = 4) – 10.2 (n = 6) – – – – – – 7.1 (n = 10)
[5] [9] [44] [45] [46] [13] [6] [14] [25] [29] this work
Table 2 Concentrations of GSH and GSSG in human whole blood and hepatoma cell HepG2 and Be17402. Blood sample 1 −1
GSH (mol L ) GSSG (mol L−1 ) GSH (mol/cell) GSSG (mol/cell) GSH/GSSG a
494.0 ± 23.6 (587.0 ± 51.0) 72.0 ± 9.8
6.8
Blood sample 2 a)
438.4 ± 23.2 (426.0 ± 8.0) 76.8 ± 8.4
HepG2 cell
Be17402 cell
(5.0 ± 0.6) × 10−9 (3.2 ± 0.3) × 10−11 156.2
(3.4 ± 0.7) × 10−9 (2.3 ± 0.4) × 10−11 147.8
a)
5.7
The data in the parenthesis of GSH in the blood samples were obtained with the HPLC method.
concentrations of GSH and GSSG in the human whole blood samples and the extract of hepatoma cells HepG2 and Be17402. The results were shown in Table 2. As a control experiment, the determination of GSH in blood samples was also performed by using the HPLC and fluorometric detector following precolumn derivatization with o-phthalaldehyde [10]. Since the concentration of GSH in cells extract was below the detection limit of the HPLC method, thus we qualified the concentration in the extract of hepatoma cells only by SERS method. As shown in Table 2, the concentrations of GSH and GSSG were in the range of 400–500 and 50–100 mol L−1 in human whole blood samples, while 3–5 and 2–3 fmol/cell in hepatoma cells HepG2 and Be17402, respectively. Because the detector integrated with our HPLC equipment was limited, we could not quantify GSSG in our laboratory. Therefore, Table 2 gave the concentrations of only GSH but not GSSG in blood samples obtained by the HPLC method. The results for the quantification of GSH obtained by our SERS method are in agreement with that by the HPLC method. Moreover, we conducted another control experiment of standard addition. By adding 300 mol L−1 GSH in to the two blood samples, the recovery of GSH was 99.0% and 106.3% from the two blood samples, further demonstrating that the proposed SERS method is qualified for the detection in real complex samples.
4. Conclusions Fe3 O4 /Ag nanoparticles with a uniform size distribution were prepared as SERS substrate. The characterization indicated that metallic Ag was uniformly deposited on the Fe3 O4 magnetic nanoparticles. CV is Raman sensitive, and the Fe3 O4 /Ag nanoparticles produced much enhanced SERS responses of CV. In comparison with CV, GSH is not a Raman sensitive compound. However, the adsorption of GSH the Fe3 O4 /Ag nanoparticles though Ag-S bonding was much stronger than that of CV though Ag-N bonding. Therefore, the pre-adsorbed CV molecules on the surface of Fe3 O4 /Ag nanoparticles were replaced by the newly added GSH molecules. This resulted in a drastic decrease in the SERS intensity of CV. By combining the Fe3 O4 /Ag substrate and the CV Raman probe, the present work proposed a facile SERS method to detect GSH at low concentrations. After optimizing the experimental conditions, the new method could detect trace GSH at concentrations in the concentration range of 50–700 nmol L−1 with a detection limit of
40 nmol L−1 . The proposed method was applied to determine GSH and GSSG in the real samples of blood and cells, giving satisfactory analytical results and showing the merits of the method such as a good sensitivity, stability and reproducibility. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 Program) (Grant No. 2012AA06A304), and the National Science Foundation of China (Grant No. 21177044 and 21377169). References [1] T.M. Bray, C.G. Taylor, Can. J. Physiol. Pharmacol. 71 (1993) 746–751. ˜ [2] M. Asensi, J. Sastre, F.V. Pallardo, A. Lloret, M. Lehner, J.G. Asuncion, J. Vina, Methods Enzymol. 299 (1999) 267–276. [3] S.Y. Zhu, X.E. Zhao, W. Zhang, Z.Y. Liu, W.J. Qi, S. Anjum, G.B. Xu, Anal. Chim. Acta 786 (2013) 111–115. [4] T. Yamaguchi, I. Katoh, S. Kurata, Eur. J. Biochem. 269 (2002) 2782–2788. [5] S. Timur, D. Odaci, A. Dincer, F. Zihnioglu, A. Telefoncu, Talanta 74 (2008) 1492–1497. [6] T.H. Chen, W.L. Tseng, Small 8 (2012) 1912–1919. [7] X.Q. Chen, Y. Zhou, X.J. Peng, J.Y. Yoon, Chem. Soc. Rev. 39 (2010) 2120–2135. [8] C. Xie, D.F. Zhong, X.Y. Chen, Anal. Chim. Acta 788 (2013) 89–98. [9] G.P. McDermott, J.M. Terry, X.A. Conlan, N.W. Barnett, P.S. Francis, Anal. Chem. 83 (2011) 6034–6039. [10] D. Tsikas, J. Sandmann, D. Holzberg, P. Pantazis, M. Raida, J.C. Frolich, Anal. Biochem. 273 (1999) 32–40. [11] Y. Wang, Y. Xie, M. Bernier, I.W. Wainer, J. Chromatogr. A 1216 (2009) 3533–3537. [12] D.B. Liu, W.W. Chen, Y. Tian, S. He, W.F. Zheng, J.S. Sun, Z. Wang, X.Y. Jiang, Adv. Healthcare Mater. 1 (2012) 90–95. [13] L.L. Liu, Q. Ma, Y. Li, Z.P. Liu, X.G. Su, Talanta 114 (2013) 243–247. [14] P.T. Lee, R.G. Compton, Electroanalysis 25 (2013) 1613–1620. [15] Y.Q. Hao, D.D. Xiong, L.Q. Wang, W.S. Chen, B.B. Zhou, Y.N. Liu, Talanta 115 (2013) 253–257. [16] Y. Wang, J. Lu, L.H. Tang, H.X. Chang, J.H. Li, Anal. Chem. 81 (2009) 9710–9715. [17] B.K. McMahon, T. Gunnlaugsson, J. Am. Chem. Soc. 134 (2012) 10725–10728. [18] B.Y. Han, J.P. Yuan, E.K. Wang, Anal. Chem. 81 (2009) 5569–5573. [19] H. Xu, M. Hepel, Anal. Chem. 83 (2011) 813–819. [20] F. Tanaka, N. Mase, C.F. Barbas III, Chem. Commun. 15 (2004) 1762–1763. [21] M.K. Fan, G.F.S. Andrade, A.G. Brolo, Anal. Chim. Acta 693 (2011) 7–25. [22] N.P.W. Pieczonka, R.F. Aroca, Chem. Soc. Rev. 37 (2008) 946–954. [23] S.F. Zong, Z.Y. Wang, H. Chen, J. Yang, Y.P. Cui, Anal. Chem. 85 (2013) 2223–2230. [24] M.L. Cheng, B.C. Tsai, J. Yang, Anal. Chim. Acta 708 (2011) 89–96. [25] M. Larsson, J. Lindgren, J. Raman Spectrosc. 36 (2005) 394–399. [26] G.G. Huang, X.X. Han, M.K. Hossain, Y. Ozaki, Anal. Chem. 81 (2009) 5881–5888. [27] Y.C. Cao, R.C. Jin, J.M. Nam, C.S. Thaxton, C.A. Mirkin, J. Am. Chem. Soc. 125 (2003) 14676–14677.
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