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Colorimetric detection of Ag+ based on CAg+C binding as a bridge between gold nanoparticles
Sensors and Actuators B 250 (2017) 641–646
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Colorimetric detection of Ag+ based on C Ag+ C binding as a bridge between gold nanoparticles Hongyan Xi a , Mengjie Cui b , Wei Li b,∗ , Zhengbo Chen a,∗ a b
Department of Chemistry, Capital Normal University, Beijing, 100048, China Institute of High-Performance Polymer, Qingdao University of Science & Technology, Qingdao, Shandong, 266042, China
a r t i c l e
i n f o
Article history: Received 7 February 2017 Received in revised form 28 April 2017 Accepted 4 May 2017 Available online 5 May 2017 Keywords: Silver ion Gold nanoparticles Colorimetric Hybridization
a b s t r a c t We have developed aptamer-functionalized gold nanoparticles (AuNPs) for the rapid, selective, and sensitive detection of Ag+ in aqueous solution. Two DNA strands are designed to functionalized on the surface of AuNPs through Au S bond. In the presence of Ag+ , aptamer1 hybridizes with the partially complementary aptamer2 with the aid of C Ag+ C bindings, which could lead to different degrees of AuNP aggregation. This process is accompanied by a color change from red to bluish-purple as observed by naked eyes. This colorimetric sensor is shown to detect the presence of as low as a 0.236 nM concentration of Ag+ . Importantly, it could be applied to complex samples without complicated sample pretreatment and sophisticated instruments. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Monitoring of heavy metal ions in aquatic ecosystems is always an important issue because these ions exert adverse effects on the environment and also on human health [1]. Ag+ ions are one of the most toxic metallic pollutants which widely exist in soil, water and even in food. It has been known that Ag+ is toxic for humans at a concentration higher than 0.9 mM in drinking water. Excessive intake of Ag+ would cause argyria in humans and silver deposition has been found in the gingival, skin, liver, cornea, and kidney of patients [2]. Therefore, monitoring trace amounts of Ag+ is highly important. Numerous traditional techniques, mainly including inductively coupled plasma-atomic emission spectroscopy (ICP-MS) [3,4], atomic absorption spectroscopy (AAS) [5], and stripping voltammetry [6], are available for Ag+ ions detection. Although these approaches have made great contributions toward Ag+ assays, these methods are highly restricted by the high-cost and nonportability of these instruments, sophisticated sample preparation, and the need of professional operation. [7,8]. Therefore, it is essential to develop simple, rapid, inexpensive, and on-site applicable detection methods which can solve the above drawbacks.
∗ Corresponding authors. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.snb.2017.05.020 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Recently, much more effort has been made to explore various methods for Ag+ detection, such as fluorescent, colorimetric, and electrochemical assays [9–17]. Among these, colorimetric technique is particularly attractive because of its advantages of simplicity, rapidity, cost effectiveness and no requirement of any sophisticated instrumentation. Moreover, the detection results can be easily observed with the naked eye [18]. gold nanoparticles (AuNPs) have attracted much attention in colorimetric detection because of their convenient synthesis in aqueous solution, high solubility in water, high molar extinction coefficient, strong photostability, tunable optical properties, and so forth [19]. Thus, AuNP-based colorimetric detection of Ag+ is more attractive. Herein, Utilizing the principles of metal-ion-mediated base pairs (C Ag C) [20–22], we have rationally designed two oligonucleotides (aptamer1 and aptamer2) with three C C mismatched bases-functionalized AuNPs for colorimetric detection of Ag+ . When Ag+ ions were present in the reaction solution, the aptamer1 and the aptamer2 hybridized and formed the double-strand DNA. Thus, the distance between AuNPs was drawn closer, leading to the aggregation of AuNPs with a concomitant red-to-bluish-purple color change. Thus, the concentrations of Ag+ could be identified by measuring the decrease of UV–vis absorption intensity. The minimum detectable Ag+ concentration of the method was up to 0.236 nM. The proposed colorimetric approach was applied successfully to determine Ag+ in tap water samples.
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Scheme 1. Schematic representation of the visual detection of Ag+ based on aptamer-functionalized AuNP aggregation.
Fig. 1. The effects of (A) AuNP concentration, (B) pH, (C) ionic strength of the solution, and (D) aptamer concentration on the absorbance of AuNPs. Error bars represent the standard deviation of triplicates.
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Fig. 2. (A) Absorbance intensity changes caused by various concentration of Ag+ (0–5 M). (B) Visualization photo of quantitative determination of Ag+ . (a → h) a: 0, b: 1 nM, c: 10 nM, d: 50 nM, e: 100 nM, f: 500 nM, g: 1 M, and h: 5 M Ag+ . (C) Effect of different Ag+ concentrations on the absorbance responses. Inset: the linear relationship between A and the logarithm of Ag+ concentration ranges from 1 nM to 1 M. The inset is the corresponding derived calibration curve. (D) The absorbance of various Ag+ concentrations measured with respect to the absorbance of the blank. Error bars represent the standard deviation of triplicates.
2. Experimental section
mission electron microscope (TEM) images were obtained on a Hitachi (H-7650, 80 kV) transmission electron microscope.
2.1. Reagents and chemicals Chloroauric acid (HAuCl4 ), trisodium citrate, and AgNO3 were purchased from Sigma-Aldrich. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Alfa Aesar. DNA oligonucleotides were synthesized by Sangon Biotechnology Inc. (Shanghai, China). Their base sequences were designed as follows: Aptamer1: 5 -SH-TCAGCCCGGC-3 , and Aptamer2: 5 SH-GCCCCCCTGA-3 were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China).All other reagents are of analytical reagent grade. All solutions were prepared with double distilled water. 5 mM phosphate buffered saline (PBS) (pH 6.2) buffer was employed as buffer and washing buffer.
2.2. Instrumentation Ultraviolet–visible (UV–vis) absorption spectra were recorded on an UV-2550 Spectrophotometer (Shimadzu Corporation). Trans-
2.3. Synthesis of Au NPs AuNPs with a diameter of 15 nm were synthesized based on the reported literature [23]. In brief, first, 250 mL of HAuCl4 solution (1 mM) was heated to 100 ◦ C. Then, 25 mL of trisodium citrate solution (38.8 mM) was quickly injected to the HAuCl4 solution. The mixed solution was stirred thoroughly and heated for another 15 min until the solution color changed from light yellow to dark red. The AuNP concentration was calculated as 10 nM according to the Beer-Lambert law [24]. 2.4. Preparation of DNA-AuNP conjugates Prior to the preparation of DNA-AuNP conjugates, two DNA strands (aptamer1 and aptamer2) were processed as follows: each DNA (1 M) including 1 mM TCEP in 5 mM phosphate-buffered saline (PBS) was heated to 85 ◦ C for 5 min, and then cooled to room
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Fig. 3. TEM images of AuNPs in the presence of (A) 0, (B) 1 nM, (C) 10 nM, (D) 0.1 M, and (E) 1 M Ag+ .
for 2 min prior to spectroscopy measurement. The corresponding maximal absorption band of AuNPs was at 525 nm. Thus, the UV–vis absorption spectra were measured over the wavelength ranging from 400 nm to 800 nm. 3. Results and discussion 3.1. Sensing mechanism
Fig. 4. Responses of the sensor to the 11 common ions. The inset shows corresponding photos. Error bars represent the standard deviation of triplicates.
temperature. Then 70 L of the treated aptamer1 and aptamer2 were separately injected to 230 L AuNP solution (3.57 nM) and kept for 12 h at 37 ◦ C. After incubation, the DNA with thiol group at one end formed a strong link with AuNPs through strong covalent bond between AuNPs and −SH. Subsequently, the free DNA existed in the supernatant were removed by intense centrifugation at 11000 rpm for about 10 min. Finally, the DNA-AuNP conjugates were dispersed in 300 L PBS. 2.5. Detection of Ag+ First, aptamer1-AuNP solution and aptamer2-AuNP solution with the same concentration were mixed at a volume ratio of 1:1. Then, different concentrations of Ag+ solution were added into the above solution. The mixture was incubated at room temperature
The principle of the two aptamers-functionalized AuNPs-based colorimetric assay was showed in Scheme 1. In the absence of Ag+ , aptamer1-AuNPs and aptamer2-AuNPs repel each other in the solution. Thus the color of the detection solution remained red. Whereas in the presence of Ag+ , the formation of C Ag+ C bindings prompted the hybridization of aptamer1and aptamer2. C Ag+ C, as a bridge linked the AuNPs, leading to the aggregation of AuNPs. The color of the detection solution would turn to bluish-purple or even blue. (Scheme 1) 3.2. Optimization of experimental conditions In order to achieve the best performance for detecting Ag+ , AuNP concentration, pH, ionic strength of the solution, and DNA concentration were investigated. The optimum of AuNP concentration was optimized by incubating different AuNP concentrations with fixed concentration of aptamers. Different concentrations of AuNPs (4.35, 3.57, 2.86 nM) were obtained by diluting the prepared AuNP solution (10 nM) to different times (2.3, 2.8, 3.5). 3.57 nM AuNP concentration showed the highest sensitivity of the sensor (Fig. 1A, Fig. S1). The pH value for the aptamer Ag+ binding was also investigated. As shown in Fig. 1B, Fig. S2, the A (A = A0 A, A0 and A stand for the absorbance obtained in the absence and presence of Ag+ , respectively) increased with the increase of the
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Fig. 5. (A) UV–visible absorption spectra of AuNPs upon the addition of Ag+ ions (0, 1, 5, 10, 50, 100, 500, 1000, 5000 nM). (B) Absorbance change of AuNPs versus the logarithmic concentrations of Ag+ ions from tap water and buffer solution samples, respectively. Error bars represent the standard deviation of triplicates.
pH value (5.8–6.2). However, with further increase the pH value, the A decreased. Thus, pH 6.2 was selected for the aptamer Ag+ binding. To investigate further the role of ionic strength of the solution on the sensitivity, the A obtained from the solutions were plotted as a function of NaH2 PO4 concentrations in Fig. 1C, Fig. S3. The result shows that maximum A was achieved when the NaH2 PO4 concentration was 5 mM. The concentration of aptamer played important roles in the detection of Ag+ . Different concentrations of aptamer (ranging from 10 nM to 1 M) were incubated with 0.1 M Ag+ . As seen in Fig. 1D, the A reached a maximum at 50 nM and then drops down. Therefore, 50 nM aptamer was selected to attain a good sensitivity for the detection of Ag+ . (Fig. 1)
3.4. The selectivity Excellent anti-interference performance is another critical issue which should be considered when using this colorimetric probe in the “real-world”. Since our sensor is fabricated by specifically Ag+ responsive aptamers, it would not response to other common metal ions. As expected, as shown in Fig. 4, none of the following metal ions including Cd2+ , Co2+ , Cu2+ , Fe3+ , Hg2+ , Mn2+ , Ni2+ , Pb2+ , Zn2+ , and NO3 − at a concentration of 10 M, produce noticeable responses to the sensor compared with that of 1 M Ag+ . Apparently, the interference of these environmentally relevant metal ions to the sensor is negligible.
3.5. Analytical application
3.3. The sensitivity Under the optimal conditions, Ag+ standards with different concentrations were monitored based on the designed colorimetric sensing strategy. The absorbance decreased with the increment of target Ag+ concentration in the solution (Fig. 2A). An obvious color change from red to bluish-purple was observed with an increasing concentration of target Ag+ (Fig. 2B), which was even seen with the naked eye. A linear relationship between the absorbance change and the logarithm of Ag+ level was fitted to the experimental points from 1 nM to 1 M (inset of Fig. 2C). The detection limit (LOD) was 0.236 nM estimated at the 3Sblank criterion (Fig. 2D), which was far lower than the threshold value of silver (∼460 nM) in drinking water permitted by the United States Environmental Protection Agency (EPA). The Ag+ induced aggregation of AuNPs was further confirmed by TEM images. As shown in Fig. 3, As expected, AuNPs were well-dispersed in the absence of Ag+ and presence of DNA though a high salt concentration of 5 mM was introduced (Fig. 3A), While in the presence of target Ag+ , AuNPs began to aggregate. Further increase in target concentration induced larger AuNP aggregates (Fig. 3(B–E)). The TEM results were consistent with the UV–vis absorption spectra as well as the color change of the solution in the absence and presence of Ag+ , thus the designed colorimetric sensor was suitable for Ag+ detection.
The application feasibility of the sensor was investigated for tap water and filtered through 0.22 M nitrocellulose membranes to remove physical impurities. Aliquots of the tap water were spiked with Ag+ at different concentrations and diluted 10 times with PBS (Fig. 5A). The relationships between the absorbance change and the logarithms of the concentrations of Ag+ from tap water and buffer solution samples were separately obtained (Fig. 5B). Accordingly, Ag+ in tap water could be quantified in the linear concentration ranges from 1 nM to 1 M. In view of the influence of numerous minerals and organics existed in tap water samples on the Ag+ detection, no significant difference existed between the measured value in buffer solution and tap water samples. The results revealed the potential applicability of this Ag+ colorimetric biosensor in real samples.
4. Conclusion In summary, a highly sensitive and selective colorimetric sensor for Ag+ detection based on AuNP aggregation induced by C Ag+ C binding. Using the designed colorimetric probe with controlled optimal aptamer concentration, we observed clear assay solution color changes from red to bluish-purple as a function of increasing Ag+ concentration. In addition to wide linearity (1nM–1 m) and low detection limit (0.236 nM), the sensor possessed a near realtime (2 min) analytical feature. The excellent sensing performance
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of the colorimetric method substantially enable its application in Ag+ detection in tap water samples. Acknowledgement All authors gratefully acknowledge the financial support of Scientific Research Project of Beijing Educational Committee (Grant No. KM201710028009). 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.snb.2017.05.020. References [1] L.M. Campbell, D.G. Dixon, R.E.J. Hecky, Toxicol. Environ. Health. B 6 (2003) 325. [2] C. Greulich, D. Braun, A. Peetsch, J. Diendorf, B. Siebers, M. Epple, M. Koller, RSC Adv. 2 (2012) 6981. [3] Y. Li, C. Chen, B. Li, J. Sun, J. Wang, Y. Gao, Y. Zhao, Z. Chai, J. Anal. At. Spectrom. 21 (2006) 94. [4] S. Jitjaicham, Express Polym. Lett. 7 (2013) 832. [5] H. Yang, Z. Zhou, F. Li, T. Yi, C. Huang, Inorg. Chem. Commun. 10 (2007) 1136. [6] R. Mikelova, J. Baloun, J. Petrlova, V. Adam, L. Havel, J. Petrek, A. Horna, R. Kizek, Bioelectrochemistry 70 (2007) 508. [7] T. Liu, G. Li, N. Zhang, Y. Chen, J. Hazard. Mater. 201-202 (2012) 155. [8] A. Kumar, V. Kumar, U. Diwan, K.K. Upadhyay, Sens. Actuators, B 176 (2013) 420. [9] Z. Lin, X. Li, H.B. Kraatz, Anal. Chem. 83 (2011) 6896. [10] W. Li, G.Y. Wu, W.J. Qu, Q. Li, J.C. Lou, Q. Lin, H. Yao, Y.M. Zhang, T.B. Wei, Sens. Actuators, B 239 (2017) 671. [11] X.L. Xing, H.X. Yang, M.L. Tao, W.Q. Zhang, J. Hazard. Mater. 297 (2015) 207. [12] K.B.A. Ahmed, S. Kumar, P.A. Veerappan, J. Lumin. 77 (2016) 228.
[13] H. Zheng, M. Yan, X.X. Fan, D. Sun, S.Y. Yang, L.J. Yang, J.D. Li, Y.B. Jiang, Chem. Commun. 48 (2012) 2243. [14] Y. Fu, L. Mu, X. Zeng, J.L. Zhao, C. Redshaw, X.L. Ni, T. Yamato, Dalton Trans. 42 (2013) 3552. [15] M.1 Wang, G.W. Meng, Q. Huang, RSC Adv. 4 (2014) 8055. [16] J.P. Ma, S.Q. Wang, C.W. Zhao, H.Y. Wang, Y.B. Dong, Chem. Commun. 50 (2014) 4721. [17] R.J. Gao, G. Xu, L.S. Zheng, Y.J. Xie, M.L. Tao, W.Q. Zhang, J. Mater. Chem. C 4 (2016) 5996. [18] J.S. Lee, M.S. Han, C.A. Mirkin, Angew. Chem. Int. Ed. 46 (2007) 4093. [19] X.J. Liu, Z.J. Wu, Q.Q. Zhang, W.F. Zhao, C.H. Zong, H.W. Gai, Anal. Chem. 88 (2016) 2119. [20] L. Zhang, Z.X. Wang, R.P. Liang, J.D. Qiu, Langmuir 29 (2013) 8929. [21] X.H. Zhou, D.M. Kong, H.X. Shen, Anal. Chim. Acta 678 (2010) 124. [22] Z.H. Qing, X.X. He, K.M. Wang, Z. Zou, X. Yang, J. Huang, G.P. Yan, Anal. Methods 4 (2012) 3320. [23] J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [24] R.C. Jin, G.S. Wu, Z. Li, C.A. Mirkin, G.C. Schatz, J. Am. Chem. Soc. 125 (2003) 1643.
Biographies Hongyan Xi is currently doing her MS work at Department of Chemistry, Capital Normal University. She is currently working toward sensors. Mengjie Cui is currently doing his MS work at Institute of High Performance Polymer, Qingdao University of Science & Technology. Wei Li received his MS degree from Qingdao University of Science & Technology and Ph.D degree from Peking University. He is currently working at Institute of High Performance Polymer, Qingdao University of Science & Technology. Zhengbo Chen received his MS degree from Beijing University of Chemical Engineering and Ph.D degree from Beihang University. He is currently working at Department of Chemistry, Capital Normal University. He studied in the area of analytical chemistry. He is currently working toward sensors.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Colorimetric detection of Ag+ based on C Ag+ C binding as a bridge between gold nanoparticles Hongyan Xi a , Mengjie Cui b , Wei Li b,∗ , Zhengbo Chen a,∗ a b
Department of Chemistry, Capital Normal University, Beijing, 100048, China Institute of High-Performance Polymer, Qingdao University of Science & Technology, Qingdao, Shandong, 266042, China
a r t i c l e
i n f o
Article history: Received 7 February 2017 Received in revised form 28 April 2017 Accepted 4 May 2017 Available online 5 May 2017 Keywords: Silver ion Gold nanoparticles Colorimetric Hybridization
a b s t r a c t We have developed aptamer-functionalized gold nanoparticles (AuNPs) for the rapid, selective, and sensitive detection of Ag+ in aqueous solution. Two DNA strands are designed to functionalized on the surface of AuNPs through Au S bond. In the presence of Ag+ , aptamer1 hybridizes with the partially complementary aptamer2 with the aid of C Ag+ C bindings, which could lead to different degrees of AuNP aggregation. This process is accompanied by a color change from red to bluish-purple as observed by naked eyes. This colorimetric sensor is shown to detect the presence of as low as a 0.236 nM concentration of Ag+ . Importantly, it could be applied to complex samples without complicated sample pretreatment and sophisticated instruments. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Monitoring of heavy metal ions in aquatic ecosystems is always an important issue because these ions exert adverse effects on the environment and also on human health [1]. Ag+ ions are one of the most toxic metallic pollutants which widely exist in soil, water and even in food. It has been known that Ag+ is toxic for humans at a concentration higher than 0.9 mM in drinking water. Excessive intake of Ag+ would cause argyria in humans and silver deposition has been found in the gingival, skin, liver, cornea, and kidney of patients [2]. Therefore, monitoring trace amounts of Ag+ is highly important. Numerous traditional techniques, mainly including inductively coupled plasma-atomic emission spectroscopy (ICP-MS) [3,4], atomic absorption spectroscopy (AAS) [5], and stripping voltammetry [6], are available for Ag+ ions detection. Although these approaches have made great contributions toward Ag+ assays, these methods are highly restricted by the high-cost and nonportability of these instruments, sophisticated sample preparation, and the need of professional operation. [7,8]. Therefore, it is essential to develop simple, rapid, inexpensive, and on-site applicable detection methods which can solve the above drawbacks.
∗ Corresponding authors. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.snb.2017.05.020 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Recently, much more effort has been made to explore various methods for Ag+ detection, such as fluorescent, colorimetric, and electrochemical assays [9–17]. Among these, colorimetric technique is particularly attractive because of its advantages of simplicity, rapidity, cost effectiveness and no requirement of any sophisticated instrumentation. Moreover, the detection results can be easily observed with the naked eye [18]. gold nanoparticles (AuNPs) have attracted much attention in colorimetric detection because of their convenient synthesis in aqueous solution, high solubility in water, high molar extinction coefficient, strong photostability, tunable optical properties, and so forth [19]. Thus, AuNP-based colorimetric detection of Ag+ is more attractive. Herein, Utilizing the principles of metal-ion-mediated base pairs (C Ag C) [20–22], we have rationally designed two oligonucleotides (aptamer1 and aptamer2) with three C C mismatched bases-functionalized AuNPs for colorimetric detection of Ag+ . When Ag+ ions were present in the reaction solution, the aptamer1 and the aptamer2 hybridized and formed the double-strand DNA. Thus, the distance between AuNPs was drawn closer, leading to the aggregation of AuNPs with a concomitant red-to-bluish-purple color change. Thus, the concentrations of Ag+ could be identified by measuring the decrease of UV–vis absorption intensity. The minimum detectable Ag+ concentration of the method was up to 0.236 nM. The proposed colorimetric approach was applied successfully to determine Ag+ in tap water samples.
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Scheme 1. Schematic representation of the visual detection of Ag+ based on aptamer-functionalized AuNP aggregation.
Fig. 1. The effects of (A) AuNP concentration, (B) pH, (C) ionic strength of the solution, and (D) aptamer concentration on the absorbance of AuNPs. Error bars represent the standard deviation of triplicates.
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Fig. 2. (A) Absorbance intensity changes caused by various concentration of Ag+ (0–5 M). (B) Visualization photo of quantitative determination of Ag+ . (a → h) a: 0, b: 1 nM, c: 10 nM, d: 50 nM, e: 100 nM, f: 500 nM, g: 1 M, and h: 5 M Ag+ . (C) Effect of different Ag+ concentrations on the absorbance responses. Inset: the linear relationship between A and the logarithm of Ag+ concentration ranges from 1 nM to 1 M. The inset is the corresponding derived calibration curve. (D) The absorbance of various Ag+ concentrations measured with respect to the absorbance of the blank. Error bars represent the standard deviation of triplicates.
2. Experimental section
mission electron microscope (TEM) images were obtained on a Hitachi (H-7650, 80 kV) transmission electron microscope.
2.1. Reagents and chemicals Chloroauric acid (HAuCl4 ), trisodium citrate, and AgNO3 were purchased from Sigma-Aldrich. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Alfa Aesar. DNA oligonucleotides were synthesized by Sangon Biotechnology Inc. (Shanghai, China). Their base sequences were designed as follows: Aptamer1: 5 -SH-TCAGCCCGGC-3 , and Aptamer2: 5 SH-GCCCCCCTGA-3 were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China).All other reagents are of analytical reagent grade. All solutions were prepared with double distilled water. 5 mM phosphate buffered saline (PBS) (pH 6.2) buffer was employed as buffer and washing buffer.
2.2. Instrumentation Ultraviolet–visible (UV–vis) absorption spectra were recorded on an UV-2550 Spectrophotometer (Shimadzu Corporation). Trans-
2.3. Synthesis of Au NPs AuNPs with a diameter of 15 nm were synthesized based on the reported literature [23]. In brief, first, 250 mL of HAuCl4 solution (1 mM) was heated to 100 ◦ C. Then, 25 mL of trisodium citrate solution (38.8 mM) was quickly injected to the HAuCl4 solution. The mixed solution was stirred thoroughly and heated for another 15 min until the solution color changed from light yellow to dark red. The AuNP concentration was calculated as 10 nM according to the Beer-Lambert law [24]. 2.4. Preparation of DNA-AuNP conjugates Prior to the preparation of DNA-AuNP conjugates, two DNA strands (aptamer1 and aptamer2) were processed as follows: each DNA (1 M) including 1 mM TCEP in 5 mM phosphate-buffered saline (PBS) was heated to 85 ◦ C for 5 min, and then cooled to room
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Fig. 3. TEM images of AuNPs in the presence of (A) 0, (B) 1 nM, (C) 10 nM, (D) 0.1 M, and (E) 1 M Ag+ .
for 2 min prior to spectroscopy measurement. The corresponding maximal absorption band of AuNPs was at 525 nm. Thus, the UV–vis absorption spectra were measured over the wavelength ranging from 400 nm to 800 nm. 3. Results and discussion 3.1. Sensing mechanism
Fig. 4. Responses of the sensor to the 11 common ions. The inset shows corresponding photos. Error bars represent the standard deviation of triplicates.
temperature. Then 70 L of the treated aptamer1 and aptamer2 were separately injected to 230 L AuNP solution (3.57 nM) and kept for 12 h at 37 ◦ C. After incubation, the DNA with thiol group at one end formed a strong link with AuNPs through strong covalent bond between AuNPs and −SH. Subsequently, the free DNA existed in the supernatant were removed by intense centrifugation at 11000 rpm for about 10 min. Finally, the DNA-AuNP conjugates were dispersed in 300 L PBS. 2.5. Detection of Ag+ First, aptamer1-AuNP solution and aptamer2-AuNP solution with the same concentration were mixed at a volume ratio of 1:1. Then, different concentrations of Ag+ solution were added into the above solution. The mixture was incubated at room temperature
The principle of the two aptamers-functionalized AuNPs-based colorimetric assay was showed in Scheme 1. In the absence of Ag+ , aptamer1-AuNPs and aptamer2-AuNPs repel each other in the solution. Thus the color of the detection solution remained red. Whereas in the presence of Ag+ , the formation of C Ag+ C bindings prompted the hybridization of aptamer1and aptamer2. C Ag+ C, as a bridge linked the AuNPs, leading to the aggregation of AuNPs. The color of the detection solution would turn to bluish-purple or even blue. (Scheme 1) 3.2. Optimization of experimental conditions In order to achieve the best performance for detecting Ag+ , AuNP concentration, pH, ionic strength of the solution, and DNA concentration were investigated. The optimum of AuNP concentration was optimized by incubating different AuNP concentrations with fixed concentration of aptamers. Different concentrations of AuNPs (4.35, 3.57, 2.86 nM) were obtained by diluting the prepared AuNP solution (10 nM) to different times (2.3, 2.8, 3.5). 3.57 nM AuNP concentration showed the highest sensitivity of the sensor (Fig. 1A, Fig. S1). The pH value for the aptamer Ag+ binding was also investigated. As shown in Fig. 1B, Fig. S2, the A (A = A0 A, A0 and A stand for the absorbance obtained in the absence and presence of Ag+ , respectively) increased with the increase of the
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Fig. 5. (A) UV–visible absorption spectra of AuNPs upon the addition of Ag+ ions (0, 1, 5, 10, 50, 100, 500, 1000, 5000 nM). (B) Absorbance change of AuNPs versus the logarithmic concentrations of Ag+ ions from tap water and buffer solution samples, respectively. Error bars represent the standard deviation of triplicates.
pH value (5.8–6.2). However, with further increase the pH value, the A decreased. Thus, pH 6.2 was selected for the aptamer Ag+ binding. To investigate further the role of ionic strength of the solution on the sensitivity, the A obtained from the solutions were plotted as a function of NaH2 PO4 concentrations in Fig. 1C, Fig. S3. The result shows that maximum A was achieved when the NaH2 PO4 concentration was 5 mM. The concentration of aptamer played important roles in the detection of Ag+ . Different concentrations of aptamer (ranging from 10 nM to 1 M) were incubated with 0.1 M Ag+ . As seen in Fig. 1D, the A reached a maximum at 50 nM and then drops down. Therefore, 50 nM aptamer was selected to attain a good sensitivity for the detection of Ag+ . (Fig. 1)
3.4. The selectivity Excellent anti-interference performance is another critical issue which should be considered when using this colorimetric probe in the “real-world”. Since our sensor is fabricated by specifically Ag+ responsive aptamers, it would not response to other common metal ions. As expected, as shown in Fig. 4, none of the following metal ions including Cd2+ , Co2+ , Cu2+ , Fe3+ , Hg2+ , Mn2+ , Ni2+ , Pb2+ , Zn2+ , and NO3 − at a concentration of 10 M, produce noticeable responses to the sensor compared with that of 1 M Ag+ . Apparently, the interference of these environmentally relevant metal ions to the sensor is negligible.
3.5. Analytical application
3.3. The sensitivity Under the optimal conditions, Ag+ standards with different concentrations were monitored based on the designed colorimetric sensing strategy. The absorbance decreased with the increment of target Ag+ concentration in the solution (Fig. 2A). An obvious color change from red to bluish-purple was observed with an increasing concentration of target Ag+ (Fig. 2B), which was even seen with the naked eye. A linear relationship between the absorbance change and the logarithm of Ag+ level was fitted to the experimental points from 1 nM to 1 M (inset of Fig. 2C). The detection limit (LOD) was 0.236 nM estimated at the 3Sblank criterion (Fig. 2D), which was far lower than the threshold value of silver (∼460 nM) in drinking water permitted by the United States Environmental Protection Agency (EPA). The Ag+ induced aggregation of AuNPs was further confirmed by TEM images. As shown in Fig. 3, As expected, AuNPs were well-dispersed in the absence of Ag+ and presence of DNA though a high salt concentration of 5 mM was introduced (Fig. 3A), While in the presence of target Ag+ , AuNPs began to aggregate. Further increase in target concentration induced larger AuNP aggregates (Fig. 3(B–E)). The TEM results were consistent with the UV–vis absorption spectra as well as the color change of the solution in the absence and presence of Ag+ , thus the designed colorimetric sensor was suitable for Ag+ detection.
The application feasibility of the sensor was investigated for tap water and filtered through 0.22 M nitrocellulose membranes to remove physical impurities. Aliquots of the tap water were spiked with Ag+ at different concentrations and diluted 10 times with PBS (Fig. 5A). The relationships between the absorbance change and the logarithms of the concentrations of Ag+ from tap water and buffer solution samples were separately obtained (Fig. 5B). Accordingly, Ag+ in tap water could be quantified in the linear concentration ranges from 1 nM to 1 M. In view of the influence of numerous minerals and organics existed in tap water samples on the Ag+ detection, no significant difference existed between the measured value in buffer solution and tap water samples. The results revealed the potential applicability of this Ag+ colorimetric biosensor in real samples.
4. Conclusion In summary, a highly sensitive and selective colorimetric sensor for Ag+ detection based on AuNP aggregation induced by C Ag+ C binding. Using the designed colorimetric probe with controlled optimal aptamer concentration, we observed clear assay solution color changes from red to bluish-purple as a function of increasing Ag+ concentration. In addition to wide linearity (1nM–1 m) and low detection limit (0.236 nM), the sensor possessed a near realtime (2 min) analytical feature. The excellent sensing performance
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Biographies Hongyan Xi is currently doing her MS work at Department of Chemistry, Capital Normal University. She is currently working toward sensors. Mengjie Cui is currently doing his MS work at Institute of High Performance Polymer, Qingdao University of Science & Technology. Wei Li received his MS degree from Qingdao University of Science & Technology and Ph.D degree from Peking University. He is currently working at Institute of High Performance Polymer, Qingdao University of Science & Technology. Zhengbo Chen received his MS degree from Beijing University of Chemical Engineering and Ph.D degree from Beihang University. He is currently working at Department of Chemistry, Capital Normal University. He studied in the area of analytical chemistry. He is currently working toward sensors.