Biosensors and Bioelectronics 59 (2014) 40–44

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Highly sensitive visual detection of copper (II) using water-soluble azide-functionalized gold nanoparticles and silver enhancement Zhen Zhang a, Wenqing Li b, Qiuling Zhao a, Ming Cheng a, Li Xu a, Xiaohong Fang a,n a b

Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Science, Beijing 100190, PR China Department of Chemistry, Wuhan University, Wuhan 430072, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 27 December 2013 Received in revised form 21 February 2014 Accepted 1 March 2014 Available online 15 March 2014

A high-sensitive method for the visual detection of copper ions in aqueous solution is developed. The method is based on copper ion-catalyzed ‘click’ reaction between the water-soluble azide-functionalized gold nanoparticles (AuNPs) and alkyne-modified glass slide. The PEG linker was employed as a stabilizing component along with the terminal azide group to keep the AuNPs stably dispersed in water without the assistance of any organic solvent. In the presence of copper ions, the AuNPs are ‘clicked’ on the slide, and the darkness of the AuNPs in the sample spot is promoted by silver enhancement process. Only a tiny amount of sample (10 μl) is needed with the detectable concentration down to 62 pM by the commonly used flatbed scanner, which is 2–3 orders of magnitude lower than those in previous reports. The selectivity relative to other potentially interfering ions and the applicability in real samples, human serum and tap water, have also been evaluated. Our method has a good potential in point-of-use applications and environment surveys. & 2014 Elsevier B.V. All rights reserved.

Keywords: Click chemistry Silver enhancement Copper ions Naked eye detection

1. Introduction Recognition and detection of copper ions (Cu2 þ ) have attracted particular attentions as copper is an essential trace element for life and excess copper is highly toxic to organisms (Brewer, 2012; Flemming and Trevors, 1989). Various strategies and technologies have been developed to detect Cu2 þ , including atomic absorption spectroscopy (Chan and Huang, 2000), voltammetric detection (Etienne et al., 2001; Qiu et al., 2011), inductively coupled plasma mass spectrometry (ICP-MS) (Djedjibegovic et al., 2012), fluorescence spectroscopy (Ma et al., 2012; Wang et al., 2014b; Yang et al., 2011; Zhou et al., 2013) and colorimetric method (Chen et al., 2013; Liu and Lu, 2007; Yao et al., 2013a). The commercial glucometer was recently used for Cu2 þ monitoring based on multi-invertase conjugated magnetic bead signal amplification labels (Su et al., 2013). Having the advantages of not using complicated instrumentations and extra light source, visual detection of copper ions with naked eye is expected to be more suitable for practical in-situ applications (Ruan et al., 2010; Shen et al., 2013; Wang et al., 2013; Yuan and Chen, 2012; Zhou et al., 2008). For visual detection, metallic nanoparticles have received considerable attention (Guo and Irudayaraj, 2011; Wei et al., 2008,

n

Corresponding author. Tel.: þ 86 10 62653083. E-mail address: [email protected] (X. Fang).

http://dx.doi.org/10.1016/j.bios.2014.03.003 0956-5663/& 2014 Elsevier B.V. All rights reserved.

2007, 2010). Among them, gold nanoparticles (AuNPs) are the most commonly used sensing materials because of the color difference between the unaggregated and aggregated AuNPs (Wei et al., 2007; Zhen et al., 2012). Two approaches have been developed to realize the Cu2 þ -dependent color change by switching between the dispersed and aggregated state of AuNPs. The first one is based on Cu2 þ specific DNAzyme (such as self-cleaving DNAzyme and ligation DNAzyme) to adjust the aggregation of DNA modified AuNPs by DNA hybridization (Liu and Lu, 2007; Wang et al., 2010). As several factors, such as salts concentration, temperature and pH value, which influence the activity of DNAzyme and DNA hybridization also interfere with Cu2 þ detection, specific control of those factors limits the application of DNAzyme sensors. The second approach to sense copper ions is based on ‘Click Chemistry’, due to the high efficiency of Cu þ catalyzed the cycloaddition reaction between azides and alkynes modified AuNPs (Hua et al., 2012; Lin et al., 2012; Zhou et al., 2008). Only a very small amount of Cu þ is needed for this reaction, resulting in reaction induced the aggregation of gold nanoparticles. The ‘click’ reaction can tolerate a wide range of solvents, ionic strength, temperatures and pH values, enabling a robust sensor under different conditions (Hua et al., 2012; Lin et al., 2012; Shen et al., 2013; Zhou et al., 2008). However, for these sensors, organic solvents are normally needed to enhance the stability of azides/ alkynes modified AuNPs (Hua et al., 2012; Lin et al., 2012; Zhou et al., 2008). Moreover, it should be noted that without washing

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processes in these sensors, visual signals are easily interfered by the sample background, thus lowering sensitivity in the analysis of biological samples. While the sensitivity of Cu2 þ could reach subnM using the fluorescence spectroscopy (Wang et al., 2014b) and electrochemical detection (Cui et al., 2014), the previous reported colorimetric sensing systems normally offer a detection limit in the μM to sub-μM range for Cu2 þ (Shen et al., 2013; Zhou et al., 2008). More sensitive colorimetric methods are still demanded for the detection of Cu2 þ in the complex aqueous samples. To achieve the ultra-sensitive visual detection, utilization of gold nanoparticle-induced silver enhancement property has been regarded as an effective strategy (Cheng et al., 2011; Taton et al., 2000; Wang et al., 2008). AuNPs can promote the deposition of silver in the presence of silver ion and a reducing reagent (e.g., hydroquinone), leading to a significantly increased size and optical absorption of AuNPs. Silver enhancement has long been used in histochemical electron microscopy studies for decades, and recently it was used to develop an ultrasensitive DNA detection method in a format of DNA array (Taton et al., 2000). The sensitivity of the scanometric DNA array which used silver enhancement exceeded that of the normal colorimetric method by three orders of magnitude (Reynolds et al., 2000; Taton et al., 2000). Since then, silver enhancement was also employed to detect mercuric ions, adenosine and so on (Hou et al., 2007; Lee and Mirkin, 2008; Wang et al., 2008; Zhang et al., 2009). The silver enhancement method has also showed a much improved sensitivity comparing to the colorimetric assays, such as 10 times higher in mercuric ions detection (Lee et al., 2007; Lee and Mirkin, 2008) and three orders higher in adenosine detection (Liu and Lu, 2006; Zhang et al., 2009). In this work, we developed a highly sensitive Cu2 þ detection method using the silver enhancement strategy to amplify the signal, and PEG modification to prepare the water-soluble AuNPs. The water soluble azide-functionalized AuNPs are linked to an alkynefunctionalized glass slide through “click” reaction, in the presence of Cu2þ and the reductant (sodium ascorbate). The AuNPs captured on the slide surface were visualized by the silver enhancement. As far as we know, we have realized the most sensitive visual detection of 62 pM Cu2þ in aqueous samples. 2. Experimental 2.1. Materials and reagents 3-Glycidoxypropyltrimethoxysilane (GPTMS), silver enhancer solution A and B were purchased from Sigma-Aldrich (USA). Acetylene-PEG4-amine and azido-PEG4-NHS ester were purchased from Click Chemistry Tools (USA). Thiol-PEG-Amine (average molecular weight of 5000) was purchased from Jenkem Technology Co., Ltd. (Beijing, China). All other chemicals, unless otherwise indicated, were of analytical grade and used without further purification. All aqueous solutions were prepared with ultrapure water (18.2 MΩ cm  1) from a Millipore Milli-Q water purification system (Millipore, MA, USA). 2.2. Instrumentation Thermo Scientific Nicolet iN10™ was used to obtain FT-IR spectroscopy for the characterization of modified AuNPs and acetylene-terminated glass slide. Dynamic light scattering (DLS) and the average diameter measurements were performed using Malvern Zetasizer Nano ZS-Model ZEN3600 (Worcester, UK). 2.3. Preparation of water-soluble azide-functionalized AuNPs AuNPs (13 nm in diameter) were synthesized with the citrate reduction method (Grabar et al., 1995). The water-soluble azide-

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functionalized AuNPs (azide-PEG-AuNPs) was prepared as following. Thiol-PEG-amine and AuNPs were mixed at room temperature (the final concentration of thiol-PEG-amine and AuNPs were 2 mM and 10 nM, respectively). After standing for 4 h, NaCl solution was added to a final concentration of 0.3 M. The solution was allowed to age for additional 24 hours. The mixture was then centrifugated to remove excess reagents and washed twice. The red precipitate was redispersed in 0.1 M carbonate buffer solution (pH 9). Then, to the above mixture, azide-PEG4-NHS ester was added and stirred overnight to obtain azide-PEG-AuNPs. Finally, the azide-PEGAuNPs were purified by repeated centrifugation and redispersion in water. 2.4. Preparation of terminal alkyne-functionalized glass slide Glass slides were first treated with piranha solution for 30 min at 95 1C, and washed three times with ultrapure water. Then they were silanized using GPTMS 1% (v/v) for 24 h at room temperature to obtain epoxy-modified slide (Cheng et al., 2010). Acetylene-PEG4-amine solution (10 μL, 0.1 mM) was spotted on the epoxy-modified slide at the defined locations to form a droplet array. Then the slide was incubated for 24 h in a humidified chamber. Finally, the slide was blocked with 1 M ethanolamine. 2.5. Visual detection of copper ion For copper ion detection, 10 μl of the solution containing 1 nM azide-PEG-AuNPs, 0.1 mM sodium ascorbate and different concentrations of copper sulfate (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM and 0 nM) was dropped onto the alkyne-functionalized slide and incubated for 1 h. After washing with water containing 0.1% SDS and drying with N2 gas, the slide was covered with silver enhancer solution for 10 min and washed with distilled water. Then the slides were scanned using an optical flatbed scanner (CanoScan LiDE 200) to obtain the optical images. The 8-bit greyscale values (averaged by the sample spot size, which was 5 70.6 mm) were obtained from histogram averages in Adobe Photoshop software (Cheng et al., 2010; Cho et al., 2012; Taton et al., 2000). 2.6. Analysis of the serum and tap water samples In general, copper ions in serum are mostly associated with proteins and enzymes (Blicharska et al., 2008; Saito et al., 2013). For Cu2 þ detection in serum, digestion is a commonly used procedure for sample pre-treatment (Lee et al., 2012; Wang et al., 2014a; Yuan and Chen, 2012). In our work, human serum sample (Sigma, USA) was firstly digested as reported (Lee et al., 2012). Briefly, 1 mL of serum was diluted with 8 mL ultrapure water and then treated with 1 mL of HNO3 for 4 h. The sample was briefly centrifuged at 4000 rpm for 2 min. The supernatant was heated to nearly dryness. The residual was redissolved in ultrapure water and heated to dryness again. It was finally dissolved in 10 mL ultrapure water and neutralized with 5 M NaOH. Then the sample was subjected to direct detection. The serum sample was also analyzed using Thermo ICP-MS XII (Thermo Fisher, USA) Tap water sample was collected from our laboratory. Briefly, aliquots of tap water samples were spiked with standard Cu2 þ solution at 0, 2  10  8, 1  10  7, 1  10  6 and 1  10  5 M, respectively. The spiked samples were mixed with azide-PEG-AuNPs and sodium ascorbate. Then 10 μl of the mixture solution was dropped onto the alkyne-functionalized slide and incubated for 1 h. After silver enhancement, the color change was observed by the naked eye and photographed with an optical flatbed scanner. The data was analyzed with an 8-bit greyscale using Image J.

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The tap water sample was also analyzed using Thermo ICP-MS XII (Thermo Fisher, USA).

3. Results and discussion 3.1. Detection strategy and characterization of azide functionalized gold nanoparticles As illustrated in Scheme 1, our method is based on the capture of AuNPs onto the solid surface by Cu2 þ dependent click reaction, followed by silver enhancement. In the presence of Cu2 þ , the reduction of Cu2 þ by sodium ascorbate resulted in the production of Cu þ , which was used to connect water-soluble azide-PEGAuNPs, to the terminal alkyne-functionalized glass slide through click reaction. Those AuNPs linked to the glass slide were visualized by silver staining amplification to achieve Cu2 þ detection. In our method, the PEG linker was employed as a stabilizing component along with the terminal azide group to keep the AuNPs stably dispersed in water without the assistance of any organic solvent. There are two possible configurations for the immobilized PEG polymer on surface. At low PEG concentrations, the polymer chain assumes a mushroom like configuration, where the polymer headgroups are buried. At high concentrations, the polymer chain extends out to form a brush configuration (Marsh et al., 2003). To make sure the azide groups were unmasked by PEG, we grafted PEG onto the AuNPs through a high-concentration of thiol-PEGamine. Dynamic light scattering (DLS) measurements showed that after the attachment of thiol-PEG-amine, the average diameter of the nanoparticles changed from 20.4 nm for the bare AuNPs to 42.7 nm for the amine-PEG-AuNPs (Table 1). Therefore, the capping thickness of PEG was 11.15 nm, which is close to both the theoretical calculation of the height of the brushed PEG (11.2 nm) (Marsh et al., 2003) and the experimental result (9.5 nm) in previous report (Manson et al., 2011). After the next modification step of changing the amine group to azide group, the diameter of AuNPs was unchanged due to the resolution limitation of DLS. Therefore, we expected that the PEG chains adopted an extended conformation on the azide-PEG-AuNPs surface, with the azide groups exposing for Cu2 þ dependent click reaction. We also measured zeta potential of the modified AuNPs to demonstrate the transforming of the functional groups on the AuNPs surface. The zeta potential of AuNPs increased from  38.4 mV before grafting (citrate-stabilized) to 24.7 mV after grafting with SH-PEGamine (Table 1). The noticeable decrease in zeta potential value after azide group modification indicated that the positively charged amine group was replaced by azide group. In order to confirm the azide functionalized gold nanoparticles, the IR spectra of the synthesized gold nanoparticles were obtained. The amine-PEG-AuNPs displayed a peak of C–H (2891 cm  1) in Fig. S1.

After changing to the azide group, a new azide peak (2110 cm  1) and a new ester peak (1647 cm  1) appeared as expected. The above results suggested that the azide group was successfully modified to the AuNPs and were exposed on the AuNPs surface for the reaction with alkyne groups on the glass slide. The water soluble azide-PEG-AuNPs could be stably dispersed in water for more than one month, ensuring the sensitive detection of Cu2 þ in water. 3.2. Detection of Cu2 þ with high sensitivity and selectivity With the well-dispersed azide-PEG-AuNPs, the possibility of the naked eye copper detection based on silver enhancement was examined. When a drop of mixture solution containing copper (II) ions, sodium ascorbate and azide-PEG-AuNPs was added on the alkyne modified glass slide to initiate the click reaction, the sample spot turned black after the introduction of silver enhancer (Fig. 1). In all control experiments with any one of the four compounds (Cu2 þ , sodium ascorbate, azide-PEG-AuNPs and silver enhancer) missing in drop, the sample spots were kept colorless. The darkness of the spot was also dependent on the duration time of click reaction. The greyscale gradually increased with the reaction time (Fig. S2). At 60 min the signal reached saturation. Thus we chose 60 min of click reaction throughout the experiments. To determine the minimum concentration of Cu2 þ detected by naked eye, different concentrations of Cu2 þ (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM and 0 nM) were added into the ‘click’ reaction

Table 1 The average diameter from dynamic light scattering and zeta potential of modified AuNPs. Sample

Z-average diameter (nm)

Zeta potential (mV)

AuNPs Amine-PEG-AuNPs Azide-PEG-AuNPs

20.4 7 0.5 42.7 7 0.2 42.7 7 0.7

 38.4 70.3 24.773.6  11.4 71.1

Fig. 1. Photographic image for copper ion detection.

Scheme 1. The naked eye detection assay of copper ions using silver enhancement of azide-PEG-AuNPs, which specifically bind to the alkyne-functionalized glass slide by click chemistry.

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mixture of azide-PEG-AuNPs and sodium ascorbate. Then 10 μl of the mixture was spotted onto the alkyne-functionalized slide. The darkness of the sample spots was dependent on the concentration of Cu2 þ (Fig. 2A), and 0.1 nM Cu2 þ could be clearly discriminated from the blank control by naked eye. Using a simple flatbed scanner, the quantitative correlation of greyscale value with the concentration of Cu2 þ is shown in Fig. 2B. The linear range is 0.1 nM–10 μM with calibration equation Greyscale¼ 301.06725.51  lgCCu. The detection limit is 62 pM on the basis of 3s/S. Due to silver enhancement magnification in our method, the detection limit is much lower than those of previously reported click chemistry based on Cu2 þ visual assays (Hua et al., 2012; Lin et al., 2012; Qu et al., 2011; Shen et al., 2013; Yao et al., 2013b; Zhou et al., 2008). For example, a colorimetric method was reported to detect Cu2 þ by click reaction between azide- and alkyne-functionalized AuNPs (Zhou et al., 2008). The aggregation of AuNPs led to the color change of the solution after 24 h with the detect limitation of 20 μM Cu2 þ and 1 μM Cu2 þ after further optimization (Qu et al., 2011). Lin et al. used electrochemical reduction of Cu2 þ via bulk electrolysis instead of using sodium ascorbate and 1 nM Cu2 þ could be detected by naked eye (Lin et al. 2012). With much less demanding on apparatus, we realized the most sensitive assay for visual copper ions detection. The selectivity of this assay for Cu2 þ was examined by detecting several other metal ions such as K þ , Na þ , Ca2 þ , Mg2 þ , Mn2 þ , Fe3 þ , Ba2 þ , Pb2 þ , Co2 þ , Zn2 þ (Fig. 3). We also detected the influence of other cations (Ag þ , Fe2 þ ) and anions (Cl  , NO3 , SO2 4 )(Fig. S3). The results showed that none of them interfere with Cu2 þ even at a 10fold higher concentration. The high selectivity was attributed to the high specificity of the alkyne-azide click reaction which is catalyzed only by copper ions. Comparing with other copper ion detection methods based on Cu2 þ chelating (Qin et al., 2010), our assay has the advantage of no need to use masking agents to remove the interference metal ions (such as Fe3 þ ) before copper detection.

3.3. Detect copper ions in human serum and tap water To explore the practical application of our assay, we analyzed copper ions in human serum and real water samples, respectively. The samples were analyzed using the calibration equation derived

Fig. 2. The sensitivity of copper ion detection by naked eye (A) Images of sample spots with different concentrations of copper ions. (B) Plot of 8-bit greyscale value of sample spot as a function of copper ion concentration.

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Fig. 3. The photographic images (A) and 8-bit greyscale value (B) of the sample solutions containing the mixtures of azide-PEG-AuNPs, sodium ascorbate and different metal cations. Concentration of K þ , Na þ , Ca2 þ , Mg2 þ , Mn2 þ , Fe3 þ , Ba2 þ , Pb2 þ , Co2 þ and Zn2 þ is 100 μM. The Cu2 þ concentration is 10 μM.

Table 2 Determination of Cu2 þ in tap water samples. Samplesa

Added (μM)

Mean foundb (μM)

Mean recoveryc (%)

RSDd (%)

1 2 3 4 5

0 0.02 0.1 1 10

0.0211 0.0417 0.121 1.08 9.32

– 91 98 106 93

2.2 2.9 2.7 1.8 6.2

a Samples were analyzed by calibration equation: Greyscale ¼301.067 25.51  lgCCu. b Mean concentration of three replicates. c Mean recovery (%) ¼100  (Cmean found  23.4 nM)/Cadded. d Relative standard deviation of three determinations.

from Fig. 2 (Greyscale ¼ 301.06 725.51  lgCCu). It is known that the levels of copper ions in serum is influenced by infection, inflammation, pregnancy, Wilson disease and cancer (Abbate et al., 2013; Kaiafa et al., 2012; Kumar et al., 2007; Liu et al., 2010). Using our new approach, we determined that the concentration of copper ions in human serum was 17.87 0.58 μM (n ¼3), which fell within the normal range (15.7–23.6 μM) and the reported data (Kuo et al. 2011). It was also agrees well with the value determined by ICP-MS (18.75 7 0.63 μM, n ¼3). This result suggested that the proposed method can be successfully applied to detect Cu2 þ ions in human serum. We then employed our method to check the concentration of Cu2 þ in laboratory tap water. The original concentration of Cu2 þ in tap water was determined to be 23.47 1.6 nM (n ¼3) by ICP-MS. Most of former reported visual Cu2 þ assays could not detect this level of Cu2 þ as it is much lower than the detection limit given by the those methods (Shen et al., 2013; Xu et al., 2010; Yuan and Chen, 2012; Zhou et al., 2008). Thus, they just analyzed the asprepared water samples spiked with Cu2 þ at high concentration levels (Shen et al., 2013; Yuan and Chen, 2012). In this work, due to the high sensitivity of our method, we were able to directly measure the Cu2 þ concentration. The concentration of Cu2 þ was determined to be 21.1 76.8 nM (n ¼3), which is a good agreement with the value that obtained by ICP-MS. To check the recoveries of

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the standard addition, the tap water was also spiked with different concentrations of copper ions and then analyzed with our assay. As shown in Table 2, the satisfactory recovery of standard addition (91–106%) was obtained. The above results demonstrated that our method offers a practical tool for the determination of Cu2 þ in complicated samples. 4. Conclusions In summary, a simple method for the naked eye detection of copper ions has been developed based on the copper catalyzed click reactions and the use of water-soluble azide-functionalized AuNPs as well as silver enhancement strategy. The assay was highly selective and exhibited a detection limit of 62 pM copper ion in water with flatbed scanner. Only a small amount of sample is needed, which is advantageous for rare sample detection. Moreover, our assay does not rely on any organic cosolvents, enzymatic magnification reactions, or complicated instrumentations, pointing toward potential point-of-use applications and environment surveys. Acknowledgments This work was supported by National Basic Research Program of China (Nos. 2013CB933701 and 2011CB911001), NSFC (Nos. 21127901 and 21121063), and Chinese Academy of Sciences. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.003. References Abbate, S., Giorgianni, C., D'Arrigo, G., Brecciaroli, R., Catanoso, R., Alibrando, C., Spatari, G., Gangemi, S., Abbate, C., 2013. Toxicol. Ind. Health 29, 737–745. Blicharska, B., Witek, M., Fornal, M., MacKay, A.L., 2008. J. Magn. Reson. 194, 41–45. Brewer, G.J., 2012. J. Trace Elem. Med. Biol. 26, 89–92. Chan, M.S., Huang, S.D., 2000. Talanta 51, 373–380. Chen, Z., Liu, R., Wang, S., Qu, C., Chen, L., Wang, Z., 2013. RSC Adv. 3, 13318–13323. Cheng, W., Chen, Y., Yan, F., Ding, L., Ding, S., Ju, H., Yin, Y., 2011. Chem. Commun. 47, 2877–2879. Cheng, W., Ding, L., Chen, Y., Yan, F., Ju, H., Yin, Y., 2010. Chem. Commun. 46, 6720–6722. Cho, H., Jung, J., Chung, B.H., 2012. Chem. Commun. 48, 7601–7603. Cui, L., Wu, J., Li, J., Ge, Y., Ju, H., 2014. Biosens. Bioelectron. 55, 272–277. Djedjibegovic, J., Larssen, T., Skrbo, A., Marjanovic, A., Sober, M., 2012. Food Chem. 131, 469–476. Etienne, M., Bessiere, J., Walcarius, A., 2001. Sens. Actuators B: Chem. 76, 531–538. Flemming, C.A., Trevors, J.T., 1989. Water Air Soil Pollut. 44, 143–158.

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