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Controlled side-by-side assembly of gold nanorods: A strategy for lead detection
Sensors and Actuators B 196 (2014) 252–259
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Controlled side-by-side assembly of gold nanorods: A strategy for lead detection Huai-Hong Cai, Dewen Lin, Jinhui Wang, Pei-Hui Yang ∗ , Jiye Cai Department of Chemistry, Jinan University, Guangzhou 510632, China
a r t i c l e
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Article history: Received 26 November 2013 Received in revised form 6 January 2014 Accepted 17 January 2014 Available online 25 January 2014 Keywords: Assembly Functionalization Gold nanorods Lead ions Sensing
a b s t r a c t A label-free and aggregation-based gold nanorods (AuNRs) probe has been developed for the detection of Pb2+ in aqueous solution, based on the fact that Pb2+ ions induce assembly mediated signal enhancement of cysteine-functionalized AuNRs. Cysteine (Cys) molecules are conjugated on AuNR surfaces to form cysteine-modified AuNRs (Cys-AuNRs), acting as nanoprobes in the detection of Pb2+ . Transmission electron microscopy (TEM) and UV–vis absorption spectroscopy data reveal the formation of controlled side-by-side assembly of the AuNRs in the presence of Pb2+ . The formation of aggregation of AuNRs significantly enhances detection signals, leading to dramatic decrease in the longitudinal surface plasmon resonance (SPR) absorption. The experiment conditions, including AuNRs aspect ratio, reaction time, pH value and salt concentration, are optimized. The Cys-AuNRs probe is highly sensitive (LOD = 0.1 nM) and selective toward Pb2+ ions, with a liner detection range from 0.1 nM to 1.0 nM. This system only becomes less sensitive when other metal ion is present at a very high concentration (i.e., >0.5 M). The cost-effective nanoprobes allow rapid and simple determination of the concentration of Pb2+ ions in city tap water samples, with results showing its practicality for the detection of lead in real samples. © 2014 Published by Elsevier B.V.
1. Introduction Contamination by heavy metal ions, particularly lead ions (Pb2+ ), poses a serious threat to human health and the environment. Lead poisoning has been related to several diseases associated with damage to the kidneys, the liver, and nervous system [1,2]. The maximum contamination level for lead in drinking water is defined by the US Environmental Protection Agency (EPA) to be 75 nM [3,4]. Because of its toxicity, the accurate determination of Pb2+ is critical. Traditional methods for Pb2+ analysis have been developed, including those based on atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry (ICPMS), and reversed-phase high-performance liquid chromatography coupled with UV–vis or fluorescence detection [5–8]. With regard to sensitivity and accuracy, these methods are efficient tools for Pb2+ determination, but they are time-consuming, expensive, and/or require sophisticated equipment. Recent years have witnessed great progress in the development of optical and electrochemical techniques for the detection of lead ions [9,10]. Procedures using oligonucleotides [11], chromophores [12], DNAzyme [13,14], functional spherical gold nanoparticles [15], and quantum dots [16], have all been developed for the
∗ Corresponding author. Tel.: +86 20 85223569; fax: +86 20 85223569. E-mail addresses: [email protected] (H.-H. Cai), [email protected] (P.-H. Yang). 0925-4005/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2014.01.062
selective detection of Pb2+ . However, many of these systems present limitations, for example, poor aqueous solubility, high synthetic cost, complicated processing, the use of unstable biomoleucles, or poor sensitivity. In response to these shortcomings, a cost-effective and simple detection method is required for rapid detection of lead ions. Gold nanorods (AuNRs) have attracted increasing interest ascribes to the unique properties arising from their anisotropy [17–19]. The two distinct plasmon bands, characteristics of AuNRs, are due to the oscillation of electrons along the transverse and longitudinal axis. AuNRs possess two plasmon absorption bands with the transverse surface plasmon resonance (SPR) band at 520 nm and the longitudinal SPR band [20]. The position of the longitudinal SPR band can be tuned as a function of the AuNRs aspect ratio (length to width ratio) from visible to near-infrared region [21]. The longitudinal SPR in AuNRs is extremely sensitive, even more than the plasmon absorption detectable in spherical gold nanoparticles, to any change in dielectric properties of the surrounding environment, including specific solvent and possible presence of adsorbate [22,23]. Indeed, assembly mediated new hybridized plasmon modes have been found arising as a function of the arrangements of the AuNRs, typically turning out in a significant shift and a concomitant broadening of the SPR band, which can be readily observed down to nanomolar concentrations by aggregation-induced signal enhancement [24–26]. So far, there are two types of aggregation characteristics for AuNRs including
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side-by-side assembly and end-to-end assembly. Therefore, AuNRs aggregation based sensors have been mainly developed based on dramatically sensitive detection signals in the longitudinal SPR. For example, an ion-directed strategy was provided to sensitively detect Hg2+ at ppt level by using end-to-end assembly of AuNRs [27]. Inspired by signal amplification from nanorods aggregation, controlled assembly of AuNRs to create stabilized nanoclusters are favorable for designing in sensitively detecting of Pb2+ . Although end-to-end assembly of AuNRs have been used in heavy metal detection [28–30], the controlled side-by-side assembly of AuNRs to create new assay for Pb2+ detection has not be demonstrated. Commonly, AuNRs are synthesized by means of methods that make them stabilized in aqueous solution by a surfactant, typically based on cetyltrimethylammonium bromide (CTAB) [31]. Considering the biotoxicity of CTAB, molecules can be used as specific ligands for functionalized conjugation on AuNR surfaces [19]. The interesting candidate molecules as AuNRs functionalizing agent is a thiol-derived amino or carboxyl ligand [32]. Such molecules, cysteine (Cys), have already been used for heavy metal detection due to heavy metal ion selective coordination [33]. Cysteine-capped AuNRs have demonstrated the ability to detect down to 10 ppb of Hg2+ [33]. In this study, the cysteine-functionalized AuNRs are demonstrated as selective Pb2+ probes with low detection limit. Synthesized AuNRs are functionalized with cysteine molecules, which are characterized by UV–vis absorption spectroscopy and transmission electron microscopy. Pb2+ detection tests have been performed, monitoring the changes in longitudinal SPR. The selectivity of this system has been tested for comparison by investigating several different heavy metal ions, including Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ ions and monitored by UV–vis absorption spectroscopy. The proposed assay represents a rapid, low cost, and convenient assay for Pb2+ ion detection without using any specific chemical reaction or complicated instruments.
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Purified AuNR solution (1.0 mL, 1.5 × 10−3 M) was dispersed in 2.0 mL of Cys aqueous solution (0.01 M), and a mixture was kept overnight under mild stirring. To remove the excess of free Cys, the Cys-functionalized AuNR solution was centrifuged twice at 5000 rpm for 10 min, and the precipitate was re-dispersed in 3 mL of PBS. 2.3. Characterizations of nanorods The AuNRs solution and detection tests of metal ions were investigated by UV–vis absorption spectroscopy using a UV-1901 UV–Vis spectrophotometer (Beijing, China) in the 400–900 nm range. The concentration of the AuNRs was determined according to Beer’s law using an extinction coefficient of longitudinal plasmon resonance max [34]. The nanostructures of AuNRs, Cys-functionalized AuNRs (CysAuNRs) and AuNRs in the presence of Pb2+ were characterized by transmission electron microscope (TEM) using a Jeol JEM-1400 microscope operating at 100 kV with an accelerating voltage of 200 kV. The samples were prepared by depositing drops of aqueous nanorod dispersions onto a carbon-coated copper grid and then allowing the aqueous solvent to evaporate. 2.4. Cys-AuNRs-based sensor for Pb2+ Before detection, the solution of Cys-AuNRs was kept under stirring for 10 min and monitored by UV–vis absorption measurements to check the stability of AuNRs in solution. Subsequently, increasing Pb2+ amounts were added to the Cys-AuNRs solution to measure the changes in absorption intensity. The interference analysis of other metal ions, such as Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ , was detected under the same experiment conditions. Reported data are presented as mean values with a standard deviation obtained from the analysis of four replicates.
2. Materials and methods
2.5. Analysis of real water samples
2.1. Materials
A water sample collected from city tap water was filtered through a 0.2 m membrane. Aliquots of the water were spiked with the standard Pb2+ solutions. The spiked samples were then analyzed by the developed sensing method.
Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 ·3H2 O, ≥99.9%), cetyltrimethylammonium bromide (CTAB, ≥96%), silver nitrate (AgNO3 , 99.9999%), l-ascorbic acid (99%), and sodium borohydride (NaBH4 , ∼99%) were purchased from Aldrich. AAS grade heavy metal standard solutions (Pb2+ , Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ ) and cysteine (Cys) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). MilliQ ultrapure water was used in each experiment (Millipore Milli-Q system). The buffer was 0.1 M PBS solution (pH 7.4). 2.2. Synthesis of AuNRs and Cys-functionalized conjugation of AuNRs Water-soluble AuNRs were synthesized using a seed-mediated method [34]. Briefly, a seed solution was prepared by mixing 5 mL of CTAB (0.2 M) and 5 mL of HAuCl4 (0.5 mM) with 0.6 mL freshly prepared ice-cold NaBH4 (10 mM). Such a solution of seeds was kept under stirring for 2 h and then used to grow AuNRs. For this purpose, 5 mL of HAuCl4 (1 mM) was dissolved in 5 mL of CTAB solution (0.2 M), in the presence of 0.1 mL of AgNO3 (4 mM), and then reduced by 70 L of ascorbic acid (0.0788 M). As the solution became colorless, a suitable amount of seed solution was added. Then, the solution turned from white to violet-brown, thus suggesting the formation of anisotropic AuNRs. The samples were purified by the excess of free surfactant by centrifugation at 10,000 rpm for 20 min.
3. Results and discussion 3.1. Functionalization of AuNRs with cysteine molecules One of the important steps for developing nanosensor is to fabricate and characterize nanoprobes that can facilitate the conjugation of functionalizing agent. UV–vis absorption spectroscopy is used to characterize spectroscopic properties of AuNRs, TEM is used to visually characterize their nanostructures. AuNRs exhibit the two typical absorption bands, transverse SPR peak at 520 nm and longitudinal SPR peak at 700 nm (Fig. 1a). Cys-conjugated AuNRs (Cys-AuNRs) show no significant changes in band shift of both plasmon bands (Fig. 1c), indicating that the exclusion of relevant particle aggregation induced by the conjugation of cysteine. The slight decrease in absorption intensity of longitudinal SPR peak, not transverse SPR peak, is observed, suggesting that cysteine molecules are mainly conjugated on the long sides of AuNR surfaces. TEM images show that individual AuNR exhibits rod and well-shared shape with the aspect ratio of 2.8 ± 0.2 (Fig. 1b). Under high-resolution TEM, the uniformly thick light band around metal rod is observed (Fig. 2a), which is ascribed to CTAB layer surrounding the nanorod surfaces [35]. Interestingly, after
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Fig. 1. (a) UV–vis absorption spectrum of CTAB-coating AuNRs. (b) TEM image of AuNRs. UV–vis absorption spectra of AuNRs and cysteine-modified AuNRs (c), and molecular structure of cysteine (d).
cysteine-functionalized conjugation on AuNR surface, TEM image shows that the appearance of the light band surrounding the metal rod looks definitely different. The difference thickness of light band on AuNR surface is ascribed to the different distribution of cysteine molecules on nanorods. Large thickness on the long sides of the rod is observed, while small thickness on the tips of the rod is existed (Fig. 2b). Such result suggests that the modification of cysteine would mainly occur at the long sides of AuNRs. It is known
that AuNRs are stabilized in aqueous solution by a bilayer of CTAB [36]. The peculiar geometry of nanorods yields higher density of CTAB at the long sides of the rods than that of the tips, thus allowing thiol-based molecules possess good affinity for Au to easily displace CTAB and then bind to the long sides of the Au nanorods (Fig. 2c). In addition, the larger surface area-to-volume ratio of long sides of nanorod renders the Au S bonding more possibly, which makes cysteine molecules likely to preferentially occupy this region. Our
Fig. 2. TEM images recorded in (a) CTAB-coating AuNRs and (b) cysteine-modified AuNRs. A sketch of a cysteine conjugation scheme on AuNR surface (c).
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Fig. 3. (a) UV–vis absorption spectra of cysteine-modified AuNRs (1.7 × 10−4 M) upon addition of increasing concentration of Pb2+ ions (from 0 to 0.6 nM). (b) TEM image of cysteine functionalized AuNRs after addition of 0.6 nM Pb2+ . On the below: sketch of the Pb2+ -mediated side-by-side assembly of AuNRs.
TEM image visually validates the modification of cysteine mainly occurs at the long sides of AuNRs, further supporting the results obtained from absorption spectra. Such result satisfactorily agrees with the decrease in absorption intensity of longitudinal SPR peak of AuNRs in the presence of cysteine.
3.2. Pb2+ detection by side-by-side assembly of AuNRs The detection of Pb2+ is then conducted in solution using CysAuNRs coupled with UV–vis absorption spectroscopy. Fig. 3a is the absorption spectra of Cys-AuNRs in the presence of different concentrations of Pb2+ . The absorbance of longitudinal SPR peak significantly decreases with the increasing concentrations of Pb2+ , while only a little change in transverse SPR peak is observed. This indicates that the linear relationship between absorbance of longitudinal SPR peak and Pb2+ concentration can be established. Furthermore, the concomitant blue-shift and broadening of the longitudinal SPR band are observed (Fig. 3a, curve c), which is ascribed to the side-by-side assembly of AuNRs [37]. TEM images further visually validate that Cys-AuNRs are assembled in a side-by-side fashion with a high degree of uniformity to produce stabilized nanoclusters predominately composed of dimmers and trimers (Fig. 3b). The occurrence of such assembly phenomenon in the presence of Pb2+ suggests that the interaction with Cys-AuNRs involves preferably their long sides, probably due to the specific topology of the cysteine in such a region of the AuNRs. The observed TEM image, the controlled side-by-side nanorods assembly, is consistent with the blue-shift of the longitudinal SPR band detected by UV–vis absorption spectra. This phenomenon is supported by the previous study of controllable side-by-side and end-to-end assembly of Au nanorods [38]. A possible mechanism of Pb2+ -mediated assembly of Cys-AuNRs is therefore provided (Fig. 3). The aggregation of CysAuNRs is reasonably accounted for the chelating reaction of Pb2+ ions with amino and carboxyl groups in cysteine [16]. Furthermore, there is the electrostatic interaction between negatively charged Cys-AuNRs and positively charged Pb2+ ions, which further pushes the self-assembly of nanorods.
An important factor in controlling the side-by-side assembly of nanorods is CTAB concentration [39]. In our experiment, CTAB concentration is fixed at 1 mM [39]. CTAB bilayer is partial desorption of at this optimal concentration, which reduces the surface charge density and the colloidal stability of AuNRs and then improves the assembly process. In addition, high surface coverage of cysteine and optimal selection of AuNRs ratio also can affect the nanorod assembly. In our experiment, this is kept at an optimal experiment condition where a controlled shift in longitudinal SPR band is observed as expected. The controlled experiments are carried out on CTAB-coating AuNRs, which are not conjugated with cysteine, by the addition of the same concentrations of Pb2+ ion solution, under the same experiment conditions used for the Cys-AuNRs. The observed absorption spectra do not show any change in the longitudinal SPR band and correspondingly absorbance even upon the addition of 10 nM of Pb2+ . Therefore, cysteine-functionalized AuNRs can be used as nanoprobes to quantitatively detect Pb2+ level. 3.3. Optimal experiment conditions To further obtain sensitive detection signals, the assay conditions, such as AuNRs aspect ratio, reaction time, pH value and salt concentration, are optimized. The aspect ratios of AuNRs affect the recognition capacity of nanoprobes, correspondingly, influence the sensitivity of this assay. In the presence of Pb2+ , short nanorods (an aspect ratio for 2.8) significantly reduce absorption intensity (Fig. 4b), while longer nanorods (an aspect ratio of 3.5) do not reduce signal obviously (Fig. 4a). This shows the detection signals of AuNRs are highly dependent on their aspect ratios. In order to enhance detection sensitivity and reduce background signal, the aspect ratio of 2.8 is chosen as the optimal aspect ratio of AuNRs for the detection of Pb2+ . It is well-known that pH can affect the detection ability of functionalized AuNRs, correspondingly, influence the intensity of detection signal. As shown in Fig. 4c, the absorbance of longitudinal SPR peak tends to be stable at the pH range of 6–7. It is known that the isoelectric point of cysteine is 5.2. When the pH value is higher
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Fig. 4. Optimal experiment conditions in this assay for (a) AuNRs aspect ratio of 3.5:1, (b) AuNRs aspect ratio of 2.8:1, (c) pH value, and (d) reaction time.
than 5.2, carboxylic acid in cysteine molecule is existed in negative charge of carboxyl group, which causes the aggregation driven by Pb2+ -mediated recognition and binding, and then induces the significant change in absorbance. In alkaline solution, when pH value is higher than 7, the low detection signal of this assay is observed, which is ascribed to the hydrolysis of metal ions. In acid solution, when pH value is lower than 4, the low detection signal of this assay is also observed, which is due to the hydrogen bond between cysteine-conjugated AuNRs. Therefore, the optimal pH range for this detection system is 6–7. The influence of reaction time between Pb2+ and Cys-AuNRs is investigated by monitoring the change in absorbance of longitudinal SPR peak. As shown in Fig. 4d, absorbance decreases initially and then tends to be stable when the reaction time is more than 20 min. This indicates that Pb2+ -induced assembly of AuNRs is stable after 20 min interaction. Therefore, 20-min time course is introduced to detect Pb2+ ions. Furthermore, the influence of salt concentration is investigated. The optimal salt concentration for this assay system is 1.0 × 10−4 M, which obtains the maximum changes in absorbance for detecting Pb2+ . 3.4. Sensitivity and selectivity for Pb2+ 3.4.1. Detection of Pb2+ Under the optimal experiment conditions, the proportional correlation of absorption intensity in longitudinal SPR peak with Pb2+ concentrations is investigated. As shown in Fig. 5a, the calibration plot exhibits a good detection range from 0.1 to 1.0 nM of Pb2+ (A = 0.502–0.0143 [Pb2+ ]; correlation coefficient, r = 0.9957), with a detection limit of 0.1 nM. Although studies for
determining Pb2+ have been reported, the detection limit of our assay system is three orders of magnitude lower than gold spherical nanoparticles-based sensors [40], and six orders of magnitude lower than the glutathione-modified AuNRs [41]. Pb2+ ions induce, besides a decrease in absorption intensity of longitudinal SPR peak, a controlled side-by-side assembly of AuNRs, thus resulting in an assembly based signal enhancement. The limit of detection of this assay is comparable or even better than those reported in the literature (Table 1). The reproducibility of this sensing system is investigated by operating eight repeated measurements of 0.4 nM Pb2+ , and the relative standard deviation (RSD) is found to be 1.16%, which suggests the reliability of this proposed method.
3.4.2. Detection of the other tested ions To verify selective performance of the Cys-AuNRs as a Pb2+ probe, the detection of other metal ions is investigated, including Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ at a concentration of 1.0 nM (Fig. 5b), under the same experiment conditions used for Pb2+ detection. Under the reaction time of 20 min for Cys-AuNRs, Pb2+ ions show a significant change in absorption intensity, while there is little change for other metal ions. This demonstrates that Cys-AuNRs can be utilized to detect Pb2+ with high sensitivity and selectivity. It is known that Pb2+ ions have high complexation constants to carboxylic groups of cysteine (log KPb = 4.1) than that of other metal ions (log KCd = 3.2) [33]. The proposed mechanism can be attributed to the coordination between Pb2+ and carboxylic or amino groups of cysteine [16], which induces the aggregation of AuNRs. We further investigated the changes in absorption intensity of Cys-AuNRs in the presence of a very high concentration of each
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Fig. 5. (a) Plots of longitude plasmon band of AuNRs upon addition of an increasing concentration of Pb2+ . Reported data are presented as mean values with a standard deviation obtained from the analysis of three replicates. (b) Decrease of absorption intensity recorded at the longitudinal SPR peak upon addition of different metal ions for the concentration of 1.0 nM. Error bars show the standard deviations of measurements taken from three independent experiments.
Table 1 Comparison of different methods for the determination of Pb2+ . Methods
Linear range (M)
Detection limit (M)
Reference
Colorimetric detection/DNAzyme Electrochemical detection/DNAzyme Colorimetric detection/gold nanoparticles Dynamic light scattering/glutathione-mediated gold nanorods
– – 0.1–10 M 0.1–0.025 mM
32 nM 0.3 M 100 nM –
[42] [14] [40] [41]
metal ion. Therefore, 0.5 M of each metal ion, 5000-fold over than 0.1 nM, is added into Cys-AuNRs as control experiments are performed. As shown in Fig. 6a, higher concentration of metal ions can induce more sensitively detection signals. Meanwhile, the change in absorbance for Hg2+ ions is noticeably observed when the concentration is higher than 0.5 M. Because Hg2+ ions have high complexation constant to cysteine [33], it is difficult to rationalize the selective response toward Pb2+ . To further evaluate the selectivity of this assay, we monitor the aggregation kinetics of Cys-AuNRs after addition of different metal ions. Plots of the time-dependent absorbance of Cys-AuNRs with 1.0 nM metal ions are obtained (Fig. 6b). Compared with other metal ions, specifically Hg2+ , Pb2+ exhibits the stronger signals in the range of 30 min. The further control experiment shows that the changes in absorbance of Cys-AuNRs upon the addition of 0.5 M Hg2+ need
about 100 min at the same conditions. Therefore, the selectivity of Cys-AuNRs for Pb2+ is partly ascribed to different aggregation rates of nanorods, which is supported by colorimetric detection of Pb2+ using glutathione-modified gold nanoparticles [40]. The detail mechanism for Pb2+ -selective detection needs further investigation. The high concentration metal ions that interfered with the detection are Hg2+ because they show a similar coordination effect as Pb2+ . This remains the limitation of our system despite its higher sensitivity and selectivity as compared to other nanoparticlesbased metal ion detection systems [41]. The Hg2+ interference is most likely due to a complex mechanism including concentrationbased effect and nanorods aggregation rate. Currently, it would be necessary to pre-treat the samples to avoid interference from Hg2+ ions in the Cys-capped AuNRs detection scheme.
Fig. 6. (a) Decrease of absorption intensity recorded at the longitudinal SPR peak upon addition of different metal ions for the concentration of 0.5 M. Error bars show the standard deviations of measurements taken from three independent experiments. (b) Plots of the time-dependent absorbance in the range of 30 min of Cys-AuNRs solution in the presence of 1.0 nM of all metal ions.
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Table 2 The application of the proposed method for analysis of real water samples with addition of different amount of Pb2+ . Sample
Water sample 1 Water sample 2 Water sample 3 Water sample 4
Concentration of Pb2+ (10−10 M) Amount added
Amount found a
1.0 2.0 3.0 4.0
1.05 1.89 3.14 3.85
± ± ± ±
0.16 0.28 0.23 0.31
Recovery (%)
105.0 94.5 104.7 96.3
Recovery = (Amount found)/(Amount added) × 100%. a Average of three determinations ± standard deviation.
Nevertheless, our system provides for simple, sensitive, and highthroughput detection for on-site screening applications prior to more vigorous analysis. 3.5. Detection of Pb2+ in real samples To test the practicality of our developed approach, we applied this assay to determine Pb2+ level in a real sample of city tap water. By applying standard addition methods to our approach, the Cys-AuNRs probe provided recoveries of 94.5–105.0% for Pb2+ ions (Table 2). Thus, our system is a practical tool for the determination of Pb2+ ions in environmental samples. 4. Conclusion In summary, we develop the assembly based signal enhancement strategy and demonstrated its application in the Pb2+ detection with a detailed analysis. The cysteine-modified AuNRs have been characterized and tested for Pb2+ detection in aqueous solution. Given the blue-shift of longitudinal SPR band specifically related to the side-by-side assembly, controlled assembly of AuNRs induced by Pb2+ can be considered to be highly selective detected, which is in comparison to the previous spherical gold nanoparticles based detection system [40]. Under optimal conditions, the CysAuNRs probe is highly sensitive (LOD = 0.1 nM) and selective toward Pb2+ ions, with a liner detection range from 0.1 nM to 1.0 nM. This system only becomes less sensitive when other metal ion is present at a very high concentration (i.e., >0.5 M). The proposed method displays good sensitivity, low detection limit and well performance in the determination of real samples without complicated modification and expensive instruments. Although the calculated limit of detection (LOD) for Pb2+ at ppb level (approximately nM) was provided by using spherical gold nanoparticles-based biosensors or chemiluminescent sensor [42], the higher sensitivity of this proposed system has been achieved without any chemical reaction or specific excitation source to activate fluorescence. In addition, the simple and general functionalized strategy of AuNRs could be effectively extended to a variety of other thiol-based derivatives, in order to screen the sensitivity and selectivity of the molecules to target metal ions. Finally, the role of the AuNRs geometry, such as, length and aspect ratio, can be further controllably designed in order to fully exploit the potential of the response to heavy metal ions. Acknowledgements This work was supported by National Natural Science Foundation China (30872404, 21375048 and 2107106), National 973 Project (2010CB833603), Specialized Research Fund for the Doctoral Program of Higher Education (20104401120004), the Fundamental Research Funds for the Central Universities (21610427 and 21612402), and the PhD Start-up Fund of Natural Science Foundation of Guangdong Province (S2012040006713).
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Biographies Huai-Hong Cai is the associate professor of Chemistry Department in Jinan University, PR China. She received her Ph.D. from Jinan University, PR China. Her research interests include bionanotechnology and bioanalytical chemistry. Dewen Lin received her B.Ac. in chemistry from Jinan University. Her research interests include analytical chemistry. Jinhui Wang received her M.Sc. in analytical chemistry from Jinan University. Her research interests include analytical chemistry. Pei-Hui Yang is the professor of Chemistry Department in Jinan University, PR China. She received her Ph.D. from Chinese Academy of Sciences, PR China. Her research interests include bioanalytical chemistry, nanomaterials and nanotechnology. Jiye Cai is the professor of Chemistry Department in Jinan University, PR China. He received his Ph.D. from Chinese Academy of Sciences (PR China) and Columbia University (USA). His research interests include bionanotechnology.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Controlled side-by-side assembly of gold nanorods: A strategy for lead detection Huai-Hong Cai, Dewen Lin, Jinhui Wang, Pei-Hui Yang ∗ , Jiye Cai Department of Chemistry, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 26 November 2013 Received in revised form 6 January 2014 Accepted 17 January 2014 Available online 25 January 2014 Keywords: Assembly Functionalization Gold nanorods Lead ions Sensing
a b s t r a c t A label-free and aggregation-based gold nanorods (AuNRs) probe has been developed for the detection of Pb2+ in aqueous solution, based on the fact that Pb2+ ions induce assembly mediated signal enhancement of cysteine-functionalized AuNRs. Cysteine (Cys) molecules are conjugated on AuNR surfaces to form cysteine-modified AuNRs (Cys-AuNRs), acting as nanoprobes in the detection of Pb2+ . Transmission electron microscopy (TEM) and UV–vis absorption spectroscopy data reveal the formation of controlled side-by-side assembly of the AuNRs in the presence of Pb2+ . The formation of aggregation of AuNRs significantly enhances detection signals, leading to dramatic decrease in the longitudinal surface plasmon resonance (SPR) absorption. The experiment conditions, including AuNRs aspect ratio, reaction time, pH value and salt concentration, are optimized. The Cys-AuNRs probe is highly sensitive (LOD = 0.1 nM) and selective toward Pb2+ ions, with a liner detection range from 0.1 nM to 1.0 nM. This system only becomes less sensitive when other metal ion is present at a very high concentration (i.e., >0.5 M). The cost-effective nanoprobes allow rapid and simple determination of the concentration of Pb2+ ions in city tap water samples, with results showing its practicality for the detection of lead in real samples. © 2014 Published by Elsevier B.V.
1. Introduction Contamination by heavy metal ions, particularly lead ions (Pb2+ ), poses a serious threat to human health and the environment. Lead poisoning has been related to several diseases associated with damage to the kidneys, the liver, and nervous system [1,2]. The maximum contamination level for lead in drinking water is defined by the US Environmental Protection Agency (EPA) to be 75 nM [3,4]. Because of its toxicity, the accurate determination of Pb2+ is critical. Traditional methods for Pb2+ analysis have been developed, including those based on atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry (ICPMS), and reversed-phase high-performance liquid chromatography coupled with UV–vis or fluorescence detection [5–8]. With regard to sensitivity and accuracy, these methods are efficient tools for Pb2+ determination, but they are time-consuming, expensive, and/or require sophisticated equipment. Recent years have witnessed great progress in the development of optical and electrochemical techniques for the detection of lead ions [9,10]. Procedures using oligonucleotides [11], chromophores [12], DNAzyme [13,14], functional spherical gold nanoparticles [15], and quantum dots [16], have all been developed for the
∗ Corresponding author. Tel.: +86 20 85223569; fax: +86 20 85223569. E-mail addresses: [email protected] (H.-H. Cai), [email protected] (P.-H. Yang). 0925-4005/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2014.01.062
selective detection of Pb2+ . However, many of these systems present limitations, for example, poor aqueous solubility, high synthetic cost, complicated processing, the use of unstable biomoleucles, or poor sensitivity. In response to these shortcomings, a cost-effective and simple detection method is required for rapid detection of lead ions. Gold nanorods (AuNRs) have attracted increasing interest ascribes to the unique properties arising from their anisotropy [17–19]. The two distinct plasmon bands, characteristics of AuNRs, are due to the oscillation of electrons along the transverse and longitudinal axis. AuNRs possess two plasmon absorption bands with the transverse surface plasmon resonance (SPR) band at 520 nm and the longitudinal SPR band [20]. The position of the longitudinal SPR band can be tuned as a function of the AuNRs aspect ratio (length to width ratio) from visible to near-infrared region [21]. The longitudinal SPR in AuNRs is extremely sensitive, even more than the plasmon absorption detectable in spherical gold nanoparticles, to any change in dielectric properties of the surrounding environment, including specific solvent and possible presence of adsorbate [22,23]. Indeed, assembly mediated new hybridized plasmon modes have been found arising as a function of the arrangements of the AuNRs, typically turning out in a significant shift and a concomitant broadening of the SPR band, which can be readily observed down to nanomolar concentrations by aggregation-induced signal enhancement [24–26]. So far, there are two types of aggregation characteristics for AuNRs including
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side-by-side assembly and end-to-end assembly. Therefore, AuNRs aggregation based sensors have been mainly developed based on dramatically sensitive detection signals in the longitudinal SPR. For example, an ion-directed strategy was provided to sensitively detect Hg2+ at ppt level by using end-to-end assembly of AuNRs [27]. Inspired by signal amplification from nanorods aggregation, controlled assembly of AuNRs to create stabilized nanoclusters are favorable for designing in sensitively detecting of Pb2+ . Although end-to-end assembly of AuNRs have been used in heavy metal detection [28–30], the controlled side-by-side assembly of AuNRs to create new assay for Pb2+ detection has not be demonstrated. Commonly, AuNRs are synthesized by means of methods that make them stabilized in aqueous solution by a surfactant, typically based on cetyltrimethylammonium bromide (CTAB) [31]. Considering the biotoxicity of CTAB, molecules can be used as specific ligands for functionalized conjugation on AuNR surfaces [19]. The interesting candidate molecules as AuNRs functionalizing agent is a thiol-derived amino or carboxyl ligand [32]. Such molecules, cysteine (Cys), have already been used for heavy metal detection due to heavy metal ion selective coordination [33]. Cysteine-capped AuNRs have demonstrated the ability to detect down to 10 ppb of Hg2+ [33]. In this study, the cysteine-functionalized AuNRs are demonstrated as selective Pb2+ probes with low detection limit. Synthesized AuNRs are functionalized with cysteine molecules, which are characterized by UV–vis absorption spectroscopy and transmission electron microscopy. Pb2+ detection tests have been performed, monitoring the changes in longitudinal SPR. The selectivity of this system has been tested for comparison by investigating several different heavy metal ions, including Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ ions and monitored by UV–vis absorption spectroscopy. The proposed assay represents a rapid, low cost, and convenient assay for Pb2+ ion detection without using any specific chemical reaction or complicated instruments.
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Purified AuNR solution (1.0 mL, 1.5 × 10−3 M) was dispersed in 2.0 mL of Cys aqueous solution (0.01 M), and a mixture was kept overnight under mild stirring. To remove the excess of free Cys, the Cys-functionalized AuNR solution was centrifuged twice at 5000 rpm for 10 min, and the precipitate was re-dispersed in 3 mL of PBS. 2.3. Characterizations of nanorods The AuNRs solution and detection tests of metal ions were investigated by UV–vis absorption spectroscopy using a UV-1901 UV–Vis spectrophotometer (Beijing, China) in the 400–900 nm range. The concentration of the AuNRs was determined according to Beer’s law using an extinction coefficient of longitudinal plasmon resonance max [34]. The nanostructures of AuNRs, Cys-functionalized AuNRs (CysAuNRs) and AuNRs in the presence of Pb2+ were characterized by transmission electron microscope (TEM) using a Jeol JEM-1400 microscope operating at 100 kV with an accelerating voltage of 200 kV. The samples were prepared by depositing drops of aqueous nanorod dispersions onto a carbon-coated copper grid and then allowing the aqueous solvent to evaporate. 2.4. Cys-AuNRs-based sensor for Pb2+ Before detection, the solution of Cys-AuNRs was kept under stirring for 10 min and monitored by UV–vis absorption measurements to check the stability of AuNRs in solution. Subsequently, increasing Pb2+ amounts were added to the Cys-AuNRs solution to measure the changes in absorption intensity. The interference analysis of other metal ions, such as Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ , was detected under the same experiment conditions. Reported data are presented as mean values with a standard deviation obtained from the analysis of four replicates.
2. Materials and methods
2.5. Analysis of real water samples
2.1. Materials
A water sample collected from city tap water was filtered through a 0.2 m membrane. Aliquots of the water were spiked with the standard Pb2+ solutions. The spiked samples were then analyzed by the developed sensing method.
Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 ·3H2 O, ≥99.9%), cetyltrimethylammonium bromide (CTAB, ≥96%), silver nitrate (AgNO3 , 99.9999%), l-ascorbic acid (99%), and sodium borohydride (NaBH4 , ∼99%) were purchased from Aldrich. AAS grade heavy metal standard solutions (Pb2+ , Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ ) and cysteine (Cys) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). MilliQ ultrapure water was used in each experiment (Millipore Milli-Q system). The buffer was 0.1 M PBS solution (pH 7.4). 2.2. Synthesis of AuNRs and Cys-functionalized conjugation of AuNRs Water-soluble AuNRs were synthesized using a seed-mediated method [34]. Briefly, a seed solution was prepared by mixing 5 mL of CTAB (0.2 M) and 5 mL of HAuCl4 (0.5 mM) with 0.6 mL freshly prepared ice-cold NaBH4 (10 mM). Such a solution of seeds was kept under stirring for 2 h and then used to grow AuNRs. For this purpose, 5 mL of HAuCl4 (1 mM) was dissolved in 5 mL of CTAB solution (0.2 M), in the presence of 0.1 mL of AgNO3 (4 mM), and then reduced by 70 L of ascorbic acid (0.0788 M). As the solution became colorless, a suitable amount of seed solution was added. Then, the solution turned from white to violet-brown, thus suggesting the formation of anisotropic AuNRs. The samples were purified by the excess of free surfactant by centrifugation at 10,000 rpm for 20 min.
3. Results and discussion 3.1. Functionalization of AuNRs with cysteine molecules One of the important steps for developing nanosensor is to fabricate and characterize nanoprobes that can facilitate the conjugation of functionalizing agent. UV–vis absorption spectroscopy is used to characterize spectroscopic properties of AuNRs, TEM is used to visually characterize their nanostructures. AuNRs exhibit the two typical absorption bands, transverse SPR peak at 520 nm and longitudinal SPR peak at 700 nm (Fig. 1a). Cys-conjugated AuNRs (Cys-AuNRs) show no significant changes in band shift of both plasmon bands (Fig. 1c), indicating that the exclusion of relevant particle aggregation induced by the conjugation of cysteine. The slight decrease in absorption intensity of longitudinal SPR peak, not transverse SPR peak, is observed, suggesting that cysteine molecules are mainly conjugated on the long sides of AuNR surfaces. TEM images show that individual AuNR exhibits rod and well-shared shape with the aspect ratio of 2.8 ± 0.2 (Fig. 1b). Under high-resolution TEM, the uniformly thick light band around metal rod is observed (Fig. 2a), which is ascribed to CTAB layer surrounding the nanorod surfaces [35]. Interestingly, after
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Fig. 1. (a) UV–vis absorption spectrum of CTAB-coating AuNRs. (b) TEM image of AuNRs. UV–vis absorption spectra of AuNRs and cysteine-modified AuNRs (c), and molecular structure of cysteine (d).
cysteine-functionalized conjugation on AuNR surface, TEM image shows that the appearance of the light band surrounding the metal rod looks definitely different. The difference thickness of light band on AuNR surface is ascribed to the different distribution of cysteine molecules on nanorods. Large thickness on the long sides of the rod is observed, while small thickness on the tips of the rod is existed (Fig. 2b). Such result suggests that the modification of cysteine would mainly occur at the long sides of AuNRs. It is known
that AuNRs are stabilized in aqueous solution by a bilayer of CTAB [36]. The peculiar geometry of nanorods yields higher density of CTAB at the long sides of the rods than that of the tips, thus allowing thiol-based molecules possess good affinity for Au to easily displace CTAB and then bind to the long sides of the Au nanorods (Fig. 2c). In addition, the larger surface area-to-volume ratio of long sides of nanorod renders the Au S bonding more possibly, which makes cysteine molecules likely to preferentially occupy this region. Our
Fig. 2. TEM images recorded in (a) CTAB-coating AuNRs and (b) cysteine-modified AuNRs. A sketch of a cysteine conjugation scheme on AuNR surface (c).
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Fig. 3. (a) UV–vis absorption spectra of cysteine-modified AuNRs (1.7 × 10−4 M) upon addition of increasing concentration of Pb2+ ions (from 0 to 0.6 nM). (b) TEM image of cysteine functionalized AuNRs after addition of 0.6 nM Pb2+ . On the below: sketch of the Pb2+ -mediated side-by-side assembly of AuNRs.
TEM image visually validates the modification of cysteine mainly occurs at the long sides of AuNRs, further supporting the results obtained from absorption spectra. Such result satisfactorily agrees with the decrease in absorption intensity of longitudinal SPR peak of AuNRs in the presence of cysteine.
3.2. Pb2+ detection by side-by-side assembly of AuNRs The detection of Pb2+ is then conducted in solution using CysAuNRs coupled with UV–vis absorption spectroscopy. Fig. 3a is the absorption spectra of Cys-AuNRs in the presence of different concentrations of Pb2+ . The absorbance of longitudinal SPR peak significantly decreases with the increasing concentrations of Pb2+ , while only a little change in transverse SPR peak is observed. This indicates that the linear relationship between absorbance of longitudinal SPR peak and Pb2+ concentration can be established. Furthermore, the concomitant blue-shift and broadening of the longitudinal SPR band are observed (Fig. 3a, curve c), which is ascribed to the side-by-side assembly of AuNRs [37]. TEM images further visually validate that Cys-AuNRs are assembled in a side-by-side fashion with a high degree of uniformity to produce stabilized nanoclusters predominately composed of dimmers and trimers (Fig. 3b). The occurrence of such assembly phenomenon in the presence of Pb2+ suggests that the interaction with Cys-AuNRs involves preferably their long sides, probably due to the specific topology of the cysteine in such a region of the AuNRs. The observed TEM image, the controlled side-by-side nanorods assembly, is consistent with the blue-shift of the longitudinal SPR band detected by UV–vis absorption spectra. This phenomenon is supported by the previous study of controllable side-by-side and end-to-end assembly of Au nanorods [38]. A possible mechanism of Pb2+ -mediated assembly of Cys-AuNRs is therefore provided (Fig. 3). The aggregation of CysAuNRs is reasonably accounted for the chelating reaction of Pb2+ ions with amino and carboxyl groups in cysteine [16]. Furthermore, there is the electrostatic interaction between negatively charged Cys-AuNRs and positively charged Pb2+ ions, which further pushes the self-assembly of nanorods.
An important factor in controlling the side-by-side assembly of nanorods is CTAB concentration [39]. In our experiment, CTAB concentration is fixed at 1 mM [39]. CTAB bilayer is partial desorption of at this optimal concentration, which reduces the surface charge density and the colloidal stability of AuNRs and then improves the assembly process. In addition, high surface coverage of cysteine and optimal selection of AuNRs ratio also can affect the nanorod assembly. In our experiment, this is kept at an optimal experiment condition where a controlled shift in longitudinal SPR band is observed as expected. The controlled experiments are carried out on CTAB-coating AuNRs, which are not conjugated with cysteine, by the addition of the same concentrations of Pb2+ ion solution, under the same experiment conditions used for the Cys-AuNRs. The observed absorption spectra do not show any change in the longitudinal SPR band and correspondingly absorbance even upon the addition of 10 nM of Pb2+ . Therefore, cysteine-functionalized AuNRs can be used as nanoprobes to quantitatively detect Pb2+ level. 3.3. Optimal experiment conditions To further obtain sensitive detection signals, the assay conditions, such as AuNRs aspect ratio, reaction time, pH value and salt concentration, are optimized. The aspect ratios of AuNRs affect the recognition capacity of nanoprobes, correspondingly, influence the sensitivity of this assay. In the presence of Pb2+ , short nanorods (an aspect ratio for 2.8) significantly reduce absorption intensity (Fig. 4b), while longer nanorods (an aspect ratio of 3.5) do not reduce signal obviously (Fig. 4a). This shows the detection signals of AuNRs are highly dependent on their aspect ratios. In order to enhance detection sensitivity and reduce background signal, the aspect ratio of 2.8 is chosen as the optimal aspect ratio of AuNRs for the detection of Pb2+ . It is well-known that pH can affect the detection ability of functionalized AuNRs, correspondingly, influence the intensity of detection signal. As shown in Fig. 4c, the absorbance of longitudinal SPR peak tends to be stable at the pH range of 6–7. It is known that the isoelectric point of cysteine is 5.2. When the pH value is higher
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Fig. 4. Optimal experiment conditions in this assay for (a) AuNRs aspect ratio of 3.5:1, (b) AuNRs aspect ratio of 2.8:1, (c) pH value, and (d) reaction time.
than 5.2, carboxylic acid in cysteine molecule is existed in negative charge of carboxyl group, which causes the aggregation driven by Pb2+ -mediated recognition and binding, and then induces the significant change in absorbance. In alkaline solution, when pH value is higher than 7, the low detection signal of this assay is observed, which is ascribed to the hydrolysis of metal ions. In acid solution, when pH value is lower than 4, the low detection signal of this assay is also observed, which is due to the hydrogen bond between cysteine-conjugated AuNRs. Therefore, the optimal pH range for this detection system is 6–7. The influence of reaction time between Pb2+ and Cys-AuNRs is investigated by monitoring the change in absorbance of longitudinal SPR peak. As shown in Fig. 4d, absorbance decreases initially and then tends to be stable when the reaction time is more than 20 min. This indicates that Pb2+ -induced assembly of AuNRs is stable after 20 min interaction. Therefore, 20-min time course is introduced to detect Pb2+ ions. Furthermore, the influence of salt concentration is investigated. The optimal salt concentration for this assay system is 1.0 × 10−4 M, which obtains the maximum changes in absorbance for detecting Pb2+ . 3.4. Sensitivity and selectivity for Pb2+ 3.4.1. Detection of Pb2+ Under the optimal experiment conditions, the proportional correlation of absorption intensity in longitudinal SPR peak with Pb2+ concentrations is investigated. As shown in Fig. 5a, the calibration plot exhibits a good detection range from 0.1 to 1.0 nM of Pb2+ (A = 0.502–0.0143 [Pb2+ ]; correlation coefficient, r = 0.9957), with a detection limit of 0.1 nM. Although studies for
determining Pb2+ have been reported, the detection limit of our assay system is three orders of magnitude lower than gold spherical nanoparticles-based sensors [40], and six orders of magnitude lower than the glutathione-modified AuNRs [41]. Pb2+ ions induce, besides a decrease in absorption intensity of longitudinal SPR peak, a controlled side-by-side assembly of AuNRs, thus resulting in an assembly based signal enhancement. The limit of detection of this assay is comparable or even better than those reported in the literature (Table 1). The reproducibility of this sensing system is investigated by operating eight repeated measurements of 0.4 nM Pb2+ , and the relative standard deviation (RSD) is found to be 1.16%, which suggests the reliability of this proposed method.
3.4.2. Detection of the other tested ions To verify selective performance of the Cys-AuNRs as a Pb2+ probe, the detection of other metal ions is investigated, including Mn2+ , Al3+ , Cr3+ , Mg2+ , Cu2+ , K+ , Zn2+ , Hg2+ , Cd2+ , and Fe3+ at a concentration of 1.0 nM (Fig. 5b), under the same experiment conditions used for Pb2+ detection. Under the reaction time of 20 min for Cys-AuNRs, Pb2+ ions show a significant change in absorption intensity, while there is little change for other metal ions. This demonstrates that Cys-AuNRs can be utilized to detect Pb2+ with high sensitivity and selectivity. It is known that Pb2+ ions have high complexation constants to carboxylic groups of cysteine (log KPb = 4.1) than that of other metal ions (log KCd = 3.2) [33]. The proposed mechanism can be attributed to the coordination between Pb2+ and carboxylic or amino groups of cysteine [16], which induces the aggregation of AuNRs. We further investigated the changes in absorption intensity of Cys-AuNRs in the presence of a very high concentration of each
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Fig. 5. (a) Plots of longitude plasmon band of AuNRs upon addition of an increasing concentration of Pb2+ . Reported data are presented as mean values with a standard deviation obtained from the analysis of three replicates. (b) Decrease of absorption intensity recorded at the longitudinal SPR peak upon addition of different metal ions for the concentration of 1.0 nM. Error bars show the standard deviations of measurements taken from three independent experiments.
Table 1 Comparison of different methods for the determination of Pb2+ . Methods
Linear range (M)
Detection limit (M)
Reference
Colorimetric detection/DNAzyme Electrochemical detection/DNAzyme Colorimetric detection/gold nanoparticles Dynamic light scattering/glutathione-mediated gold nanorods
– – 0.1–10 M 0.1–0.025 mM
32 nM 0.3 M 100 nM –
[42] [14] [40] [41]
metal ion. Therefore, 0.5 M of each metal ion, 5000-fold over than 0.1 nM, is added into Cys-AuNRs as control experiments are performed. As shown in Fig. 6a, higher concentration of metal ions can induce more sensitively detection signals. Meanwhile, the change in absorbance for Hg2+ ions is noticeably observed when the concentration is higher than 0.5 M. Because Hg2+ ions have high complexation constant to cysteine [33], it is difficult to rationalize the selective response toward Pb2+ . To further evaluate the selectivity of this assay, we monitor the aggregation kinetics of Cys-AuNRs after addition of different metal ions. Plots of the time-dependent absorbance of Cys-AuNRs with 1.0 nM metal ions are obtained (Fig. 6b). Compared with other metal ions, specifically Hg2+ , Pb2+ exhibits the stronger signals in the range of 30 min. The further control experiment shows that the changes in absorbance of Cys-AuNRs upon the addition of 0.5 M Hg2+ need
about 100 min at the same conditions. Therefore, the selectivity of Cys-AuNRs for Pb2+ is partly ascribed to different aggregation rates of nanorods, which is supported by colorimetric detection of Pb2+ using glutathione-modified gold nanoparticles [40]. The detail mechanism for Pb2+ -selective detection needs further investigation. The high concentration metal ions that interfered with the detection are Hg2+ because they show a similar coordination effect as Pb2+ . This remains the limitation of our system despite its higher sensitivity and selectivity as compared to other nanoparticlesbased metal ion detection systems [41]. The Hg2+ interference is most likely due to a complex mechanism including concentrationbased effect and nanorods aggregation rate. Currently, it would be necessary to pre-treat the samples to avoid interference from Hg2+ ions in the Cys-capped AuNRs detection scheme.
Fig. 6. (a) Decrease of absorption intensity recorded at the longitudinal SPR peak upon addition of different metal ions for the concentration of 0.5 M. Error bars show the standard deviations of measurements taken from three independent experiments. (b) Plots of the time-dependent absorbance in the range of 30 min of Cys-AuNRs solution in the presence of 1.0 nM of all metal ions.
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Table 2 The application of the proposed method for analysis of real water samples with addition of different amount of Pb2+ . Sample
Water sample 1 Water sample 2 Water sample 3 Water sample 4
Concentration of Pb2+ (10−10 M) Amount added
Amount found a
1.0 2.0 3.0 4.0
1.05 1.89 3.14 3.85
± ± ± ±
0.16 0.28 0.23 0.31
Recovery (%)
105.0 94.5 104.7 96.3
Recovery = (Amount found)/(Amount added) × 100%. a Average of three determinations ± standard deviation.
Nevertheless, our system provides for simple, sensitive, and highthroughput detection for on-site screening applications prior to more vigorous analysis. 3.5. Detection of Pb2+ in real samples To test the practicality of our developed approach, we applied this assay to determine Pb2+ level in a real sample of city tap water. By applying standard addition methods to our approach, the Cys-AuNRs probe provided recoveries of 94.5–105.0% for Pb2+ ions (Table 2). Thus, our system is a practical tool for the determination of Pb2+ ions in environmental samples. 4. Conclusion In summary, we develop the assembly based signal enhancement strategy and demonstrated its application in the Pb2+ detection with a detailed analysis. The cysteine-modified AuNRs have been characterized and tested for Pb2+ detection in aqueous solution. Given the blue-shift of longitudinal SPR band specifically related to the side-by-side assembly, controlled assembly of AuNRs induced by Pb2+ can be considered to be highly selective detected, which is in comparison to the previous spherical gold nanoparticles based detection system [40]. Under optimal conditions, the CysAuNRs probe is highly sensitive (LOD = 0.1 nM) and selective toward Pb2+ ions, with a liner detection range from 0.1 nM to 1.0 nM. This system only becomes less sensitive when other metal ion is present at a very high concentration (i.e., >0.5 M). The proposed method displays good sensitivity, low detection limit and well performance in the determination of real samples without complicated modification and expensive instruments. Although the calculated limit of detection (LOD) for Pb2+ at ppb level (approximately nM) was provided by using spherical gold nanoparticles-based biosensors or chemiluminescent sensor [42], the higher sensitivity of this proposed system has been achieved without any chemical reaction or specific excitation source to activate fluorescence. In addition, the simple and general functionalized strategy of AuNRs could be effectively extended to a variety of other thiol-based derivatives, in order to screen the sensitivity and selectivity of the molecules to target metal ions. Finally, the role of the AuNRs geometry, such as, length and aspect ratio, can be further controllably designed in order to fully exploit the potential of the response to heavy metal ions. Acknowledgements This work was supported by National Natural Science Foundation China (30872404, 21375048 and 2107106), National 973 Project (2010CB833603), Specialized Research Fund for the Doctoral Program of Higher Education (20104401120004), the Fundamental Research Funds for the Central Universities (21610427 and 21612402), and the PhD Start-up Fund of Natural Science Foundation of Guangdong Province (S2012040006713).
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Biographies Huai-Hong Cai is the associate professor of Chemistry Department in Jinan University, PR China. She received her Ph.D. from Jinan University, PR China. Her research interests include bionanotechnology and bioanalytical chemistry. Dewen Lin received her B.Ac. in chemistry from Jinan University. Her research interests include analytical chemistry. Jinhui Wang received her M.Sc. in analytical chemistry from Jinan University. Her research interests include analytical chemistry. Pei-Hui Yang is the professor of Chemistry Department in Jinan University, PR China. She received her Ph.D. from Chinese Academy of Sciences, PR China. Her research interests include bioanalytical chemistry, nanomaterials and nanotechnology. Jiye Cai is the professor of Chemistry Department in Jinan University, PR China. He received his Ph.D. from Chinese Academy of Sciences (PR China) and Columbia University (USA). His research interests include bionanotechnology.