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A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides
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A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides Fangyuan Wang, Yongxin Li, Wenying Li, Jian Chen, Qingfeng Zhang, Sohail Anjum Shahzad, Cong Yu
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Received date: 13 June 2014 Revised date: 22 August 2014 Accepted date: 27 August 2014 Cite this article as: Fangyuan Wang, Yongxin Li, Wenying Li, Jian Chen, Qingfeng Zhang, Sohail Anjum Shahzad, Cong Yu, A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.08.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides
Fangyuan Wanga,b, Yongxin Lia, Wenying Lia,b, Jian Chena, Qingfeng Zhanga,b, Sohail Anjum Shahzada,c, Cong Yu*a,b
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
c
Department of Chemistry, COMSATS Institute of Information Technology,
Abbottabad, 22060, Pakistan
*Corresponding author: Fax: +86-431-85262710 E-mail: [email protected]
1
Abstract In this work, a fluorescence turn-on method for copper(II) detection is reported. A molecular beacon (MB) was designed as a template. Cu2+ was reduced to Cu+ in the presence of a reductant (ascorbic acid). Two short single-stranded oligonucleotides one was labeled with a 5'-alkyne and the other with 3'-azide group, proceeded a template-dependent chemical ligation through the Cu(I)-catalyzed azide-alkyne cycloaddition. The newly generated click-ligated long chain oligonucleotide, which was complementary to the MB, opened the MB hairpin structure and resulted in a turn on fluorescence. The increase in fluorescence intensity is directly proportional to the amount of Cu2+ added to the assay solution. The present assay is quite sensitive and allows the detection of 2 nM Cu2+. The described assay also exhibits high selectivity over other metal ions.
Keywords: copper; click chemistry; fluorescence; molecular beacon
2
1. Introduction Copper is one of the most abundant transition metals in the human body and is essential for human health [1]. Copper plays important biological roles as enzyme cofactors and as structural components of proteins [1-3]. The lack of copper in the human body may cause copper deficiency diseases, such as coeliac disease and anaemia [4]. However, high concentration of copper is also harmful to the human body, which may cause gastrointestinal disturbance [5], liver or kidney damage [6-7], and various neurological diseases, such as Alzheimer’s disease and Parkinson’s disease [8-9]. In recent years, continuous release of copper(II) into environmental water has been witnessed due to its extensive use in industry [5]. Therefore, selective and sensitive detection of copper(II) is of great importance.
Great efforts have been made for the development of techniques for selective copper(II) sensing. They include absorption spectrometry [10], atomic absorption spectrometry [11], inductively coupled plasma atomic emission spectroscopy [12], inductively coupled plasma mass spectrometry [13], and various electrochemical [14-15], colorimetric [16-19], and fluorescent methods [20-23]. Fluorescent methods have attracted more and more attentions because of their high sensitivity, simple operation, good reproducibility, and low-cost. Although a number of fluorescent sensors for copper(II) detection have been reported, simple, sensitive and selective methods without the use of harmful substances are still highly demanded. 3
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction reported by Sharpless in 2001 [24] has emerged as one of the most prominent click chemistry reactions. And it has been used as a versatile, highly efficient and selective chemical tool in many fields [25-26]. It also possesses characters of high-speed and simplicity under mild conditions which earned its name “click reaction” [27]. A trace amount of Cu(I) could catalyze the Huisgen 1,3-dipolar cycloaddition reaction between an alkyne and an azide to form a 1,2,3-triazole [25], and the overall percentage of conversion is related to the amount of Cu(I) added to the reaction mixture. Based on the reduction reaction of Cu(II) to Cu(I) in the presence of a reductant, a number of methods for copper(II) sensing were proposed [28-34]. Recently, a few copper(II) sensors based on template-dependent oligonucleotide ligation were developed [35-39]. It has been shown that the CuAAC driven click reaction enables the ligation of oligonucleotides without any biological enzymes and it shows good biocompatibility [40-44]. However, these methods require the use of additional signaling unit, such as gold nanopartiles, graphene or organic dye, thus the reaction could not be monitored in a real-time fashion, and some used fluorescent turn-off sensing strategy which may increase the possibility of false positive signals [36-39].
Molecular beacon (MB) was originally designed for the sensing of oligonucleotides, nucleic acid point mutations, RNA targets in vivo, and for monitoring PCR reactions in real time [45]. The method shows high sensitivity and selectivity. Since its first 4
report, MB has also been used for the development of a number of novel fluorescence turn-on assays for the sensing of nucleic acid binding protein, gene expression in living cells, enzyme activity, and many aptamer targeted proteins and small biomolecules, etc. [46-51].
Herein, we describe a novel fluorescence turn-on strategy for copper(II) detection based on the template-dependent click ligation (Scheme 1). In the present method, a rationally designed MB (Oligo-MB) was adopted as a template. Two short oligonucleotides (Oligo-A and Oligo-B) labeled with a 5'-alkyne and a 3'-azide groups, respectively, were used. Oligo-A and Oligo-B could hybridize to the complementary sequences of the Oligo-MB, which brought the azide and the alkyne functional groups closer to each other. In the presence of Cu2+ and ascorbic acid, Cu2+ was reduced to Cu+, and the two short oligonucleotides were ligated by the formation of a triazole linkage. The Cu(I) catalyzed click reaction between the azide and the alkyne functional groups resulted in the connection of Oligo-A and Oligo-B. The newly formed long chain oligonucleotide (Oligo-A-B) opened the Oligo-MB hairpin structure and turn on fluorescence was therefore observed. The increase in intensity could be directly related to the amount of Cu(II) added to the assay solution. Our Cu2+ detection method is sensitive, selective, simple, and inexpensive.
5
2. Experimental Section 2.1 Materials The oligonucleotides (Oligo-A, Oligo-B and Oligo-MB) and L-ascorbic acid were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). Cupric sulfate pentahydrate (CuSO4·5H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All other chemicals were of analytical reagent grade and used without further purification. The ultra pure water used in all experiments was obtained with a Milli-Q system (Millipore, MA, USA).
2.2 Instrumentation Fluorescence measurements were performed using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc., USA). Sample solutions were excited at 480 nm and the fluorescence emission spectra were recorded with slits for excitation and emission both of 5 nm. The quantification of nucleic acids was carried out via UV-Vis absorption recorded on a Cary 50 Bio Spectrophotometer (Varian Inc., CA, USA). A PB-10 pH meter (Sartorius Scientific Instrument Co., Ltd. Beijing) was used to adjust the pH value of all buffer solutions.
2.3 Assay procedures Oligonucleotide stock solutions were prepared with Tris-HCl buffer (20 mM, pH 7.4) and stored at 4 ºC. Prior to the addition of different concentrations of Cu2+ at room temperature, the oligonucleotides and ascorbic acid were mixed and stored at 4 ºC for 6
10 min. The assay solutions were incubated at 25 ºC for an appropriate period of time before the fluorescence measurement. The final concentrations of Oligo-A, Oligo-B and Oligo-MB were 30 nM, 30 nM and 10 nM, respectively.
2.4 Lake water sample analysis Lake water samples were acquired from the South Lake of Changchun, Jilin province, China. The samples were first centrifuged at 12,000 rpm for 10 min and filtered through a nitrocellulose filter (0.45 m) to get rid of the insoluble materials. 100 L lake water sample was added into the assay solution for the quantitative analysis (final sample volume: 400 L). Known quantities of Cu2+ were added to the assay solutions and their recoveries were determined. The experiments were repeated three times and the average recovery values were obtained.
3. Results and Discussion 3.1 The assay strategy The sequences of the oligonucleotides for Cu2+ detection were carefully selected (Table 1). Oligo-MB with a fluorophore and a quencher at its termini was selected as a template. It was designed to be a stable hairpin structure at an ambient temperature. When Oligo-MB was in its hairpin state, the fluorophore and the quencher interacted with each other and the fluorescence of the fluorophore was quenched [45]. Oligo-A and Oligo-B (each of 11 nt long) were complementary to Oligo-MB and could bind to Oligo-MB. Since they were relatively short oligonucleotides, their binding could not 7
open the MB hairpin structure. However, their binding allowed to bring azide and alkyne groups labeled on Oligo-A and Oligo-B, respectively, more closer to each other, which effectively increased the local molarity of the reaction groups. The close proximity of Oligo-A and Oilgo-B and subsequently increase in local molarity enhanced the rate of click reaction. The newly formed longer Oligo-A-B through efficient click reaction could easily open the Oligo-MB hairpin structure and a turn on fluorescence was observed.
3.2 Feasibility of the assay The melting temperature of Oligo-A and Oligo-B was approximately 23-24 ºC. Oligo-MB has a melting temperature of 43 ºC (Fig. 1). When the oligonucleotides were mixed and incubated for 120 min, the fluorescence intensity of the assay mixture did not increase. The results suggest that Oligo-A and Oligo-B could not open the Oligo-MB hairpin structure. However, when 50 nM Cu2+ was introduced, Cu2+ was reduced to Cu+. Click reaction-assisted formation of Oligo-A-B stimulated the unfolding of Oligo-MB and subsequent gradual increase in the fluorescence intensity was observed with the prolonged reaction time, and the fluorescence intensity reached a plateau after about 120 min of reaction (Fig. 2). The results clearly demonstrate that our strategy for Cu2+ sensing is valid.
3.3 Optimization of assay conditions To achieve the best sensing performance, important experimental conditions were 8
optimized. In order to investigate the temperature effect on assay solution, we used various temperatures to probe the sensing performance as shown in Fig. 3. There was a rapid increase in the emission intensity as the solution temperature raised from 10 ºC to 25 ºC. It seems that the ligation efficiency increased at a higher temperature. However, if the assay temperature was higher than 30 ºC, considerable amounts of Oligo-A and Oligo-B would dissociate from Oligo-MB which resulted in decreased ligation efficiency. Therefore, 25 ºC was chosen as the reaction temperature in the following experiments. The concentration of ascorbic acid added to the assay solution was also optimized. We have experimented various concentrations of ascorbic acid as shown in Fig. 4. It was found that 500 M ascorbic acid gave the best sensing performance. The assay buffer pH was selected at 7.4 since it was reported that at acidic pH values, the Cu+ catalytic activity may be reduced because of the disproportion reaction [36,39].
3.4 Sensitivity of the assay Under the optimized conditions, we tested the sensitivity of the assay by adding different concentrations of Cu2+. Fig. 5 shows gradual increase in the fluorescence intensity with the increasing concentration of Cu2+ from 0 nM to 50 nM, and the fluorescence intensity reached a plateau at about 50 nM Cu2+. The linear detection range is 2 nM 40 nM, and the linear calibration curve is: I = 2.73C + 9.77 (I and C refer to the fluorescence intensity and the concentration of Cu2+ in nM, respectively). The method is highly sensitive, 2 nM Cu2+ could be easily detected [16-23,35-39], 9
which is much lower than the maximum allowed level of Cu2+ (20 M, 1.3 ppm) in drinking water defined by the United States Environmental Protection Agency.
3.5 Selectivity of the assay To evaluate the selectivity of the assay, other common metal ions including Ba2+, Mg2+, Ni2+, Co2+, Fe2+, Cd2+, Ca2+, Zn2+, Mn2+, Hg2+, Pb2+, Li+, Na+, K+, and Ag+ were tested. As shown in Fig. 6, the results indicate that the proposed method is highly selective for Cu2+. None of these metal ions interfered with the assay, even at much higher concentrations (50 M).
3.6 Cu2+ assay in environmental water samples To demonstrate the potential practical application of the assay for real sample analysis, lake water samples were tested. The water samples were acquired from the South Lake of Changchun (Jilin province). The samples were first centrifuged at 12,000 rpm for 10 min and filtered through a nitrocellulose filter (0.45 m) to get rid of the insoluble materials. Recovery experiments were carried out. Different concentrations of Cu2+ (20 nM, 40 nM) were added to the assay solutions, and satisfactory recovery values were obtained. The results clearly show that our method could be used for the detection of Cu2+ in complex sample mixtures (Table 2).
4. Conclusions In conclusion, a copper(II) ion sensing method with high selectivity and sensitivity 10
has been developed. The concentration of Cu2+ in an aqueous buffer solution could be determined by the molecular beacon fluorescence recovery assay. 2 nM Cu2+ could be easily detected, and a linear response range of 2 nM 40 nM Cu2+ was obtained. The fluorescent turn-on Cu2+ sensing strategy would reduce the possibility of false positive caused by the turn-off mode. And it could also be used for the analysis of environmental water samples. The method is simple, sensitive and selective, and it could potentially be used for the analysis of real biological samples because of the good biocompatibility and low toxicity.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21275139), the National Basic Research Program of China (973 Program, Grant 2011CB911002), and the Open Project of the State Key Laboratory of Supramolecular Structure and Materials (Grant No. SKLSSM201415).
References [1]
E.L. Que, D.W. Domaille, C.J. Chang, Metals in neurobiology: Probing their chemistry and biology with molecular imaging, Chem. Rev. 108 (2008) 1517-1549.
[2]
B.-E. Kim, T. Nevitt, D.J. Thiele, Mechanisms for copper acquisition, distribution and regulation, Nat. Chem. Biol. 4 (2008) 176-185.
[3]
A.K. Boal, A.C. Rosenzweig, Structural biology of copper trafficking, Chem. 11
Rev. 109 (2009) 4760-4779. [4]
M. Bonham, J.M. O’Connor, B.M. Hannigan, J.J. Strain, The immune system as a physiological indicator of marginal copper status, Brit. J. Nutr. 87 (2002) 393-403.
[5]
P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, Environmental copper: Its dynamics and human exposure issues, J. Toxicol. Env. Health, B 4 (2001) 341-394.
[6]
S.H. Hahn, M.S. Tanner, D.M. Danke, W.A. Gahl, Normal metallothionein synthesis in fibroblasts obtained from children with Indian childhood cirrhosis or copper-associated childhood cirrhosis, Biochem. Mol. Med. 54 (1995) 142-145.
[7]
B.P. Zietz, J.D. de Vergara, H. Dunkelberg, Copper concentrations in tap water and possible effects on infant's health—Results of a study in Lower Saxony, Germany, Environ. Res. 92 (2003) 129-138.
[8]
D.R. Brown, H. Kozlowski, Biological inorganic and bioinorganic chemistry of neurodegeneration based on prion and Alzheimer diseases, Dalton Trans. 13 (2004) 1907-1917.
[9]
K.J. Barnham, A.I. Bush, Metals in Alzheimer’s and Parkinson’s diseases, Curr. Opin. Chem. Biol. 12 (2008) 222-228.
[10] M.R. Callahan, J.B. Rose, R.H. Byrne, Long pathlength absorbance spectroscopy: Trace copper analysis using a 4.4 m liquid core waveguide, Talanta 58 (2002) 891-898. [11] J.R. Chen, K.C. Teo, Determination of cadmium, copper, lead and zinc in water 12
samples by flame atomic absorption spectrometry after cloud point extraction, Anal. Chim. Acta 450 (2001) 215-222. [12] O. Abollino, M. Aceto, M.C. Bruzzoniti, E. Mentasti, C. Sarzanini, Speciation of copper and manganese in milk by solid-phase extraction/inductively coupled plasma-atomic emission spectrometry, Anal. Chim. Acta 375 (1998) 299-306. [13] A.T. Townsend, K.A. Miller, S. McLean, S. Aldous, The determination of copper, zinc, cadmium and lead in urine by high resolution ICP-MS, J. Anal. At. Spectrom. 13 (1998) 1213-1219. [14] W. Yantasee, K. Hongsirikarn, C.L. Warner, D. Choi, T. Sangvanich, M.B. Toloczko, M.G. Warner, G.E. Fryxell, R.S. Addleman, C. Timchalk, Direct detection of Pb in urine and Cd, Pb, Cu, and Ag in natural waters using electrochemical sensors immobilized with DMSA functionalized magnetic nanoparticles, Analyst 133 (2008) 348-355. [15] S.Y. Qiu, L.D. Xie, S. Gao, Q.D. Liu, Z.Y. Lin, B. Qiu, G.N. Chen, Determination of copper(II) in the dairy product by an electrochemical sensor based on click chemistry, Anal. Chim. Acta 707 (2011) 57-61. [16] Z.Y. Yao, B.H. Huang, X.P. Hu, L. Zhang, D.P. Li, M. Guo, X.H. Zhang, H. Yuan, H.-C. Wu, Colorimetric detection of copper ions based on a supramolecular complex of water-soluble polythiophene and ATP, Analyst 138 (2013) 1649-1652. [17] Q.L. Xu, K.M. Lee, F. Wang, J. Yoon, Visual detection of copper ions based on azide- and alkyne-functionalized polydiacetylene vesicles, J. Mater. Chem. 21 (2011) 15214-15217. 13
[18] C.-H. Lu, Y.-W. Wang, S.-L. Ye, G.-N. Chen, H.-H. Yang, Ultrasensitive detection of Cu2+ with the naked eye and application in immunoassays, NPG Asia Mater. 4 (2012) 1884-4057. [19] X.R He, H.B. Liu, Y.L. Li, S. Wang, Y.J. Li, N. Wang, J.C. Xiao, X.H. Xu, D.B. Zhu, Gold nanoparticle-based fluorometric and colorimetric sensing of copper(II) ions, Adv. Mater. 17 (2005) 2811-2815. [20] J.H. Huang, Q.B. Zheng, J.-K. Kim, Z.G. Li, A molecular beacon and graphene oxide-based fluorescent biosensor for Cu2+ detection, Biosens. Bioelectron. 43 (2013) 379-383. [21] J.W. Liu, Y, Lu, A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity, J. Am. Chem. Soc. 129 (2007) 9838-9839. [22] H.X. Qin, J.T. Ren, J.H. Wang, E.K. Wang, G-quadruplex facilitated turn-off fluorescent chemosensor for selective detection of cupric ion, Chem. Commun. 46 (2010) 7385-7387. [23] A.W. Zhu, Q. Qu, X.L. Shao, B. Kong, Y. Tian, Carbon-dot-based dual-emission nanohybrid produces a ratiometric fluorescent sensor for in vivo imaging of cellular copper ions, Angew. Chem. Int. Ed. 51 (2012) 7185-7189. [24] H.C. Kolb, M.G. Finn, K.B. Sharpless, Click chemistry: Diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021. [25] M. Meldal, C.W. Tornøe, Cu-catalyzed azide-alkyne cycloaddition, Chem. Rev. 108 (2008) 2952-3015. 14
[26] F. Amblard, J.H. Cho, R.F. Schinazi, The Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide and oligonucleotide chemistry, Chem. Rev. 109 (2009) 4207-4220. [27] T. Efthymiou, W. Gong, J.-P. Desaulniers, Chemical architecture and applications of nucleic acid derivatives containing 1, 2, 3-triazole functionalities synthesized via click chemistry, Molecules 17 (2012) 12665-12703. [28] Y. Zhou, S.X. Wang, K. Zhang, X.Y. Jiang, Visual detection of copper (II) by azide-and alkyne-functionalized gold nanoparticles using click chemistry, Angew. Chem. Int. Ed. 47 (2008) 7454-7456. [29] C. Hua, W.H. Zhang, S.R.M. De Almeida, S. Ciampi, D. Gloria, G.Z. Liu, J.B. Harper, J.J. Gooding, A novel route to copper(II) detection using ‘click’ chemistry-induced aggregation of gold nanoparticles, Analyst 137 (2012) 82-86. [30] S. Park, H.-J. Kim, Highly selective and sensitive fluorescence turn-on probe for a catalytic amount of Cu(I) ions in water through the click reaction, Tetrahedron Lett. 53 (2012) 4473-4475. [31] Z.Y. Lin, S. Gao, J. Lin, W.L. Lin, S.Y. Qiu, L.H. Guo, B. Qiu, G.N. Chen, Visual detection of copper(II) based on the aggregation of gold nano-particles via click chemistry, Anal. Methods-UK 4 (2012) 612-615. [32] A. Lee, J. Chin, O.K. Park, H. Chung, J.W. Kim, S.-Y. Yoon, K. Park, A novel near-infrared fluorescence chemosensor for copper ion detection using click ligation and energy transfer, Chem. Commun. 49 (2013) 5969-5971. [33] J. Su, J. Xu, Y. Chen, Y. Xiang, R. Yuan, Y.Q. Chai, Sensitive detection of 15
copper(II) by a commercial glucometer using click chemistry, Biosens. Bioelectron. 45 (2013) 219-222. [34] T. Sanji, M. Nakamura, M. Tanaka, Fluorescence ‘turn-on’ detection of Cu2+ ions with aggregation-induced emission-active tetraphenylethene based on click chemistry, Tetrahedron Lett. 52 (2011) 3283-3286. [35] X.Y. Xu, W.L. Daniel, W. Wei, C.A. Mirkin, Colorimetric Cu2+ detection using DNA-modified gold-nanoparticle aggregates as probes and click chemistry, Small 6 (2010) 623-626. [36] Q.P. Shen, W.H. Li, S.Y. Tang, Y.F. Hu, Z. Nie, Y. Huang, S.Z. Yao, A simple ‘‘clickable’’ biosensor for colorimetric detection of copper(II) ions based on unmodified gold nanoparticles, Biosens. Bioelectron. 41 (2013) 663-668. [37] L.F. Zhou, Q.P. Shen, P. Zhao, B.B. Xiang, Z. Nie, Y. Huang, S.Z. Yao, Fluorescent detection of copper(II) based on DNA-templated click chemistry and graphene oxide, Methods 64 (2013) 299-304. [38] Q.P. Shen, S.Y. Tang, W.H. Li, Z. Nie, Z.L. Liu, Y. Huang, S.Z. Yao, A novel DNA-templated click chemistry strategy for fluorescent detection of copper(II) ions, Chem. Commun. 48 (2012) 281-283. [39] Q.P. Shen, L.F. Zhou, Y.J. Yuan, Y. Huang, B.B. Xiang, C.Y. Chen, Z. Nie, S.Z.Yao, Intra-molecular G-quadruplex structure generated by DNA-templated click chemistry: “Turn-on” fluorescent probe for copper ions, Biosens. Bioelectron. 55 (2014) 187-194. [40] A. H. El-Sagheer, T. Brown, Click chemistry with DNA, Chem. Soc. Rev. 39 16
(2010) 1388-1405. [41] H.B. Sun, X.H. Peng, Template-directed fluorogenic oligonucleotide ligation using “click” chemistry: Detection of single nucleotide polymorphism in the human p53 tumor suppressor gene, Bioconjugate Chem. 24 (2013) 1226-1234. [42] R. Kumar, A. El-Sagheer, J. Tumpane, P. Lincoln, L.M. Wilhelmsson, T. Brown, Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry, J. Am. Chem. Soc. 129 (2007) 6859-6864. [43] D.S. del Amo, W. Wang, H. Jiang, C. Besanceney, A.C. Yan, M. Levy, Y. Liu, F.L. Marlow, P. Wu, Biocompatible copper (I) catalysts for in vivo imaging of glycans, J. Am. Chem. Soc. 132 (2010) 16893-16899. [44] A.H. El-Sagheer, T. Brown, Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility, Chem. Sci. 5 (2014) 253-259. [45] S. Tyagi, F.R. Kramer, Molecular beacons: Probes that fluoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303-308. [46] K.M. Wang, Z.W. Tang, C.Y.J. Yang, Y.M. Kim, X.H. Fang, W. Li, Y.R. Wu, C.D. Medley, Z.H. Cao, J. Li, P. Colon, H. Lin, W.H. Tan, Molecular engineering of DNA: Molecular beacons, Angew. Chem. Int. Ed. 48 (2009) 856-870. [47] Z.W. Tang, P. Liu, C.B. Ma, X.H. Yang, K.M. Wang, W.H. Tan, X.Y. Lv, Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase, 17
Anal. Chem. 83 (2011) 2505-2510. [48] Z.W. Tang, K.M. Wang, W.H. Tan, C.B. Ma, J. Li, L.F. Liu, Q.P. Guo X.X. Meng, Real-time investigation of nucleic acids phosphorylation process using molecular beacons, Nucleic Acids Res. 33 (2005) e97. [49] F. Chen, Y.X. Zhao, L. Qi, C.H. Fan, One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification Biosens. Bioelectron. 47 (2013) 218-224. [50] Y. Cheng, J.P. Lei, Y.L. Chen, H.X. Ju, Highly selective detection of
microRNA based on distance-dependent electrochemiluminescence resonance energy transfer between CdTe nanocrystals and Au nanoclusters, Biosens. Bioelectron. 51 (2014) 431-436. [51] A.A. Goulko, F. Li, X.C. Le, Bioanalytical applications of aptamer and molecular-beacon probes in fluorescence-affinity assays, Trend Anal. Chem. 28 (2009) 878-892.
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Figure captions Scheme 1. Schematic representation of the copper(II) sensing strategy.
Table 1 The oligonucleotides employed in the current investigation.
Fig. 1 Melting temperature determination of Oligo-MB in 20 mM Tris-HCl (pH 7.4). The solution was equilibrated for 5 min prior to the fluorescence measurement. Data points were taken at 3 ºC intervals. Fluorescence intensity changes were recorded at 514 nm.
Fig. 2 a) Oligo-MB emission spectral changes with reaction time; b) Changes in fluorescence intensity with reaction time with (red) and without (black) the addition of Cu2+. The concentrations of ascorbic acid and Cu2+ were 500 M and 50 nM, respectively. Buffer: 20 mM Tris-HCl (pH 7.4). The emission intensity was recorded at 514 nm at 25 ºC.
Fig. 3 a) Fluorescence intensity changes with reaction time at different reaction temperatures. b) Enhancement of fluorescence intensity after 60 min of reaction at different temperatures. The concentrations of ascorbic acid and Cu2+ were 500 M 19
and 50 nM, respectively. Buffer: 20 mM Tris-HCl (pH 7.4). Emission intensity was recorded at 514 nm.
Fig. 4 Effect of the concentration of ascorbic acid on the performance of the assay. All the solutions were incubated with 50 nM Cu2+ at 25 ºC for 120 min in 20 mM Tris-HCl (pH 7.4). The experiments were repeated three times and the average intensity values were given.
Fig. 5 Changes in emission intensity with Cu2+ concentration (0, 2 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM). The solutions were incubated at 25 ºC for 120 min in 20 mM Tris-HCl (pH 7.4). The concentration of ascorbic acid was 500 M. The experiments were repeated three times and the average intensity values were given.
Fig. 6 Selectivity of the assay. The concentration of Cu2+ was 50 nM and the concentrations of the other metal ions (Ba2+, Mg2+, Ni2+, Co2+, Fe2+, Cd2+, Ca2+, Zn2+, Mn2+, Hg2+, Pb2+, Li+, Na+, K+, Ag+) were 50 M each. The experiments were repeated three times and the average intensity values were given. *Ag+ formed precipitate with Cl- which was removed by centrifugation prior to analysis. If the precipitate was not removed, it caused slight interference with the assay.
Table 2 Copper(II) recovery in lake water samples. 20
Scheme 1.
Table 1 Oligonucleotide sequence (5' 3') Oligo-A
alkyne-GAA ACT GAT AG
Oligo-B
CTA AAT TCC AA-azide
Oligo-MB
FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-Dabcyl
Oligo-A-B
CTA AAT TCC AA-ligated-G AAA CTG ATA G
21
Fig. 1
Fig. 2 22
Fig. 3
23
Fig. 4
Fig. 5
Fig. 6
24
Table 2 Found
Recovery
RSDa
(nM)
(nM)
(%)
(%, n=3)
1
20.0
20.6 ± 0.6
103.0
2.9
2
40.0
39.5 ± 0.9
98.8
2.3
Samples Added
a
RSD: relative standard deviation.
Highlights z
A copper(II) detection method with good sensitivity and selectivity
z
The click reaction could be monitored in a real-time fashion
z
A molecular beacon was used as the template
z
A fluorescence turn-on strategy reduces the possibility of false positive signals
25
A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides Fangyuan Wang, Yongxin Li, Wenying Li, Jian Chen, Qingfeng Zhang, Sohail Anjum Shahzad, Cong Yu
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(14)00745-0 http://dx.doi.org/10.1016/j.talanta.2014.08.064 TAL15075
To appear in:
Talanta
Received date: 13 June 2014 Revised date: 22 August 2014 Accepted date: 27 August 2014 Cite this article as: Fangyuan Wang, Yongxin Li, Wenying Li, Jian Chen, Qingfeng Zhang, Sohail Anjum Shahzad, Cong Yu, A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.08.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides
Fangyuan Wanga,b, Yongxin Lia, Wenying Lia,b, Jian Chena, Qingfeng Zhanga,b, Sohail Anjum Shahzada,c, Cong Yu*a,b
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
c
Department of Chemistry, COMSATS Institute of Information Technology,
Abbottabad, 22060, Pakistan
*Corresponding author: Fax: +86-431-85262710 E-mail: [email protected]
1
Abstract In this work, a fluorescence turn-on method for copper(II) detection is reported. A molecular beacon (MB) was designed as a template. Cu2+ was reduced to Cu+ in the presence of a reductant (ascorbic acid). Two short single-stranded oligonucleotides one was labeled with a 5'-alkyne and the other with 3'-azide group, proceeded a template-dependent chemical ligation through the Cu(I)-catalyzed azide-alkyne cycloaddition. The newly generated click-ligated long chain oligonucleotide, which was complementary to the MB, opened the MB hairpin structure and resulted in a turn on fluorescence. The increase in fluorescence intensity is directly proportional to the amount of Cu2+ added to the assay solution. The present assay is quite sensitive and allows the detection of 2 nM Cu2+. The described assay also exhibits high selectivity over other metal ions.
Keywords: copper; click chemistry; fluorescence; molecular beacon
2
1. Introduction Copper is one of the most abundant transition metals in the human body and is essential for human health [1]. Copper plays important biological roles as enzyme cofactors and as structural components of proteins [1-3]. The lack of copper in the human body may cause copper deficiency diseases, such as coeliac disease and anaemia [4]. However, high concentration of copper is also harmful to the human body, which may cause gastrointestinal disturbance [5], liver or kidney damage [6-7], and various neurological diseases, such as Alzheimer’s disease and Parkinson’s disease [8-9]. In recent years, continuous release of copper(II) into environmental water has been witnessed due to its extensive use in industry [5]. Therefore, selective and sensitive detection of copper(II) is of great importance.
Great efforts have been made for the development of techniques for selective copper(II) sensing. They include absorption spectrometry [10], atomic absorption spectrometry [11], inductively coupled plasma atomic emission spectroscopy [12], inductively coupled plasma mass spectrometry [13], and various electrochemical [14-15], colorimetric [16-19], and fluorescent methods [20-23]. Fluorescent methods have attracted more and more attentions because of their high sensitivity, simple operation, good reproducibility, and low-cost. Although a number of fluorescent sensors for copper(II) detection have been reported, simple, sensitive and selective methods without the use of harmful substances are still highly demanded. 3
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction reported by Sharpless in 2001 [24] has emerged as one of the most prominent click chemistry reactions. And it has been used as a versatile, highly efficient and selective chemical tool in many fields [25-26]. It also possesses characters of high-speed and simplicity under mild conditions which earned its name “click reaction” [27]. A trace amount of Cu(I) could catalyze the Huisgen 1,3-dipolar cycloaddition reaction between an alkyne and an azide to form a 1,2,3-triazole [25], and the overall percentage of conversion is related to the amount of Cu(I) added to the reaction mixture. Based on the reduction reaction of Cu(II) to Cu(I) in the presence of a reductant, a number of methods for copper(II) sensing were proposed [28-34]. Recently, a few copper(II) sensors based on template-dependent oligonucleotide ligation were developed [35-39]. It has been shown that the CuAAC driven click reaction enables the ligation of oligonucleotides without any biological enzymes and it shows good biocompatibility [40-44]. However, these methods require the use of additional signaling unit, such as gold nanopartiles, graphene or organic dye, thus the reaction could not be monitored in a real-time fashion, and some used fluorescent turn-off sensing strategy which may increase the possibility of false positive signals [36-39].
Molecular beacon (MB) was originally designed for the sensing of oligonucleotides, nucleic acid point mutations, RNA targets in vivo, and for monitoring PCR reactions in real time [45]. The method shows high sensitivity and selectivity. Since its first 4
report, MB has also been used for the development of a number of novel fluorescence turn-on assays for the sensing of nucleic acid binding protein, gene expression in living cells, enzyme activity, and many aptamer targeted proteins and small biomolecules, etc. [46-51].
Herein, we describe a novel fluorescence turn-on strategy for copper(II) detection based on the template-dependent click ligation (Scheme 1). In the present method, a rationally designed MB (Oligo-MB) was adopted as a template. Two short oligonucleotides (Oligo-A and Oligo-B) labeled with a 5'-alkyne and a 3'-azide groups, respectively, were used. Oligo-A and Oligo-B could hybridize to the complementary sequences of the Oligo-MB, which brought the azide and the alkyne functional groups closer to each other. In the presence of Cu2+ and ascorbic acid, Cu2+ was reduced to Cu+, and the two short oligonucleotides were ligated by the formation of a triazole linkage. The Cu(I) catalyzed click reaction between the azide and the alkyne functional groups resulted in the connection of Oligo-A and Oligo-B. The newly formed long chain oligonucleotide (Oligo-A-B) opened the Oligo-MB hairpin structure and turn on fluorescence was therefore observed. The increase in intensity could be directly related to the amount of Cu(II) added to the assay solution. Our Cu2+ detection method is sensitive, selective, simple, and inexpensive.
5
2. Experimental Section 2.1 Materials The oligonucleotides (Oligo-A, Oligo-B and Oligo-MB) and L-ascorbic acid were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). Cupric sulfate pentahydrate (CuSO4·5H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All other chemicals were of analytical reagent grade and used without further purification. The ultra pure water used in all experiments was obtained with a Milli-Q system (Millipore, MA, USA).
2.2 Instrumentation Fluorescence measurements were performed using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc., USA). Sample solutions were excited at 480 nm and the fluorescence emission spectra were recorded with slits for excitation and emission both of 5 nm. The quantification of nucleic acids was carried out via UV-Vis absorption recorded on a Cary 50 Bio Spectrophotometer (Varian Inc., CA, USA). A PB-10 pH meter (Sartorius Scientific Instrument Co., Ltd. Beijing) was used to adjust the pH value of all buffer solutions.
2.3 Assay procedures Oligonucleotide stock solutions were prepared with Tris-HCl buffer (20 mM, pH 7.4) and stored at 4 ºC. Prior to the addition of different concentrations of Cu2+ at room temperature, the oligonucleotides and ascorbic acid were mixed and stored at 4 ºC for 6
10 min. The assay solutions were incubated at 25 ºC for an appropriate period of time before the fluorescence measurement. The final concentrations of Oligo-A, Oligo-B and Oligo-MB were 30 nM, 30 nM and 10 nM, respectively.
2.4 Lake water sample analysis Lake water samples were acquired from the South Lake of Changchun, Jilin province, China. The samples were first centrifuged at 12,000 rpm for 10 min and filtered through a nitrocellulose filter (0.45 m) to get rid of the insoluble materials. 100 L lake water sample was added into the assay solution for the quantitative analysis (final sample volume: 400 L). Known quantities of Cu2+ were added to the assay solutions and their recoveries were determined. The experiments were repeated three times and the average recovery values were obtained.
3. Results and Discussion 3.1 The assay strategy The sequences of the oligonucleotides for Cu2+ detection were carefully selected (Table 1). Oligo-MB with a fluorophore and a quencher at its termini was selected as a template. It was designed to be a stable hairpin structure at an ambient temperature. When Oligo-MB was in its hairpin state, the fluorophore and the quencher interacted with each other and the fluorescence of the fluorophore was quenched [45]. Oligo-A and Oligo-B (each of 11 nt long) were complementary to Oligo-MB and could bind to Oligo-MB. Since they were relatively short oligonucleotides, their binding could not 7
open the MB hairpin structure. However, their binding allowed to bring azide and alkyne groups labeled on Oligo-A and Oligo-B, respectively, more closer to each other, which effectively increased the local molarity of the reaction groups. The close proximity of Oligo-A and Oilgo-B and subsequently increase in local molarity enhanced the rate of click reaction. The newly formed longer Oligo-A-B through efficient click reaction could easily open the Oligo-MB hairpin structure and a turn on fluorescence was observed.
3.2 Feasibility of the assay The melting temperature of Oligo-A and Oligo-B was approximately 23-24 ºC. Oligo-MB has a melting temperature of 43 ºC (Fig. 1). When the oligonucleotides were mixed and incubated for 120 min, the fluorescence intensity of the assay mixture did not increase. The results suggest that Oligo-A and Oligo-B could not open the Oligo-MB hairpin structure. However, when 50 nM Cu2+ was introduced, Cu2+ was reduced to Cu+. Click reaction-assisted formation of Oligo-A-B stimulated the unfolding of Oligo-MB and subsequent gradual increase in the fluorescence intensity was observed with the prolonged reaction time, and the fluorescence intensity reached a plateau after about 120 min of reaction (Fig. 2). The results clearly demonstrate that our strategy for Cu2+ sensing is valid.
3.3 Optimization of assay conditions To achieve the best sensing performance, important experimental conditions were 8
optimized. In order to investigate the temperature effect on assay solution, we used various temperatures to probe the sensing performance as shown in Fig. 3. There was a rapid increase in the emission intensity as the solution temperature raised from 10 ºC to 25 ºC. It seems that the ligation efficiency increased at a higher temperature. However, if the assay temperature was higher than 30 ºC, considerable amounts of Oligo-A and Oligo-B would dissociate from Oligo-MB which resulted in decreased ligation efficiency. Therefore, 25 ºC was chosen as the reaction temperature in the following experiments. The concentration of ascorbic acid added to the assay solution was also optimized. We have experimented various concentrations of ascorbic acid as shown in Fig. 4. It was found that 500 M ascorbic acid gave the best sensing performance. The assay buffer pH was selected at 7.4 since it was reported that at acidic pH values, the Cu+ catalytic activity may be reduced because of the disproportion reaction [36,39].
3.4 Sensitivity of the assay Under the optimized conditions, we tested the sensitivity of the assay by adding different concentrations of Cu2+. Fig. 5 shows gradual increase in the fluorescence intensity with the increasing concentration of Cu2+ from 0 nM to 50 nM, and the fluorescence intensity reached a plateau at about 50 nM Cu2+. The linear detection range is 2 nM 40 nM, and the linear calibration curve is: I = 2.73C + 9.77 (I and C refer to the fluorescence intensity and the concentration of Cu2+ in nM, respectively). The method is highly sensitive, 2 nM Cu2+ could be easily detected [16-23,35-39], 9
which is much lower than the maximum allowed level of Cu2+ (20 M, 1.3 ppm) in drinking water defined by the United States Environmental Protection Agency.
3.5 Selectivity of the assay To evaluate the selectivity of the assay, other common metal ions including Ba2+, Mg2+, Ni2+, Co2+, Fe2+, Cd2+, Ca2+, Zn2+, Mn2+, Hg2+, Pb2+, Li+, Na+, K+, and Ag+ were tested. As shown in Fig. 6, the results indicate that the proposed method is highly selective for Cu2+. None of these metal ions interfered with the assay, even at much higher concentrations (50 M).
3.6 Cu2+ assay in environmental water samples To demonstrate the potential practical application of the assay for real sample analysis, lake water samples were tested. The water samples were acquired from the South Lake of Changchun (Jilin province). The samples were first centrifuged at 12,000 rpm for 10 min and filtered through a nitrocellulose filter (0.45 m) to get rid of the insoluble materials. Recovery experiments were carried out. Different concentrations of Cu2+ (20 nM, 40 nM) were added to the assay solutions, and satisfactory recovery values were obtained. The results clearly show that our method could be used for the detection of Cu2+ in complex sample mixtures (Table 2).
4. Conclusions In conclusion, a copper(II) ion sensing method with high selectivity and sensitivity 10
has been developed. The concentration of Cu2+ in an aqueous buffer solution could be determined by the molecular beacon fluorescence recovery assay. 2 nM Cu2+ could be easily detected, and a linear response range of 2 nM 40 nM Cu2+ was obtained. The fluorescent turn-on Cu2+ sensing strategy would reduce the possibility of false positive caused by the turn-off mode. And it could also be used for the analysis of environmental water samples. The method is simple, sensitive and selective, and it could potentially be used for the analysis of real biological samples because of the good biocompatibility and low toxicity.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21275139), the National Basic Research Program of China (973 Program, Grant 2011CB911002), and the Open Project of the State Key Laboratory of Supramolecular Structure and Materials (Grant No. SKLSSM201415).
References [1]
E.L. Que, D.W. Domaille, C.J. Chang, Metals in neurobiology: Probing their chemistry and biology with molecular imaging, Chem. Rev. 108 (2008) 1517-1549.
[2]
B.-E. Kim, T. Nevitt, D.J. Thiele, Mechanisms for copper acquisition, distribution and regulation, Nat. Chem. Biol. 4 (2008) 176-185.
[3]
A.K. Boal, A.C. Rosenzweig, Structural biology of copper trafficking, Chem. 11
Rev. 109 (2009) 4760-4779. [4]
M. Bonham, J.M. O’Connor, B.M. Hannigan, J.J. Strain, The immune system as a physiological indicator of marginal copper status, Brit. J. Nutr. 87 (2002) 393-403.
[5]
P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, Environmental copper: Its dynamics and human exposure issues, J. Toxicol. Env. Health, B 4 (2001) 341-394.
[6]
S.H. Hahn, M.S. Tanner, D.M. Danke, W.A. Gahl, Normal metallothionein synthesis in fibroblasts obtained from children with Indian childhood cirrhosis or copper-associated childhood cirrhosis, Biochem. Mol. Med. 54 (1995) 142-145.
[7]
B.P. Zietz, J.D. de Vergara, H. Dunkelberg, Copper concentrations in tap water and possible effects on infant's health—Results of a study in Lower Saxony, Germany, Environ. Res. 92 (2003) 129-138.
[8]
D.R. Brown, H. Kozlowski, Biological inorganic and bioinorganic chemistry of neurodegeneration based on prion and Alzheimer diseases, Dalton Trans. 13 (2004) 1907-1917.
[9]
K.J. Barnham, A.I. Bush, Metals in Alzheimer’s and Parkinson’s diseases, Curr. Opin. Chem. Biol. 12 (2008) 222-228.
[10] M.R. Callahan, J.B. Rose, R.H. Byrne, Long pathlength absorbance spectroscopy: Trace copper analysis using a 4.4 m liquid core waveguide, Talanta 58 (2002) 891-898. [11] J.R. Chen, K.C. Teo, Determination of cadmium, copper, lead and zinc in water 12
samples by flame atomic absorption spectrometry after cloud point extraction, Anal. Chim. Acta 450 (2001) 215-222. [12] O. Abollino, M. Aceto, M.C. Bruzzoniti, E. Mentasti, C. Sarzanini, Speciation of copper and manganese in milk by solid-phase extraction/inductively coupled plasma-atomic emission spectrometry, Anal. Chim. Acta 375 (1998) 299-306. [13] A.T. Townsend, K.A. Miller, S. McLean, S. Aldous, The determination of copper, zinc, cadmium and lead in urine by high resolution ICP-MS, J. Anal. At. Spectrom. 13 (1998) 1213-1219. [14] W. Yantasee, K. Hongsirikarn, C.L. Warner, D. Choi, T. Sangvanich, M.B. Toloczko, M.G. Warner, G.E. Fryxell, R.S. Addleman, C. Timchalk, Direct detection of Pb in urine and Cd, Pb, Cu, and Ag in natural waters using electrochemical sensors immobilized with DMSA functionalized magnetic nanoparticles, Analyst 133 (2008) 348-355. [15] S.Y. Qiu, L.D. Xie, S. Gao, Q.D. Liu, Z.Y. Lin, B. Qiu, G.N. Chen, Determination of copper(II) in the dairy product by an electrochemical sensor based on click chemistry, Anal. Chim. Acta 707 (2011) 57-61. [16] Z.Y. Yao, B.H. Huang, X.P. Hu, L. Zhang, D.P. Li, M. Guo, X.H. Zhang, H. Yuan, H.-C. Wu, Colorimetric detection of copper ions based on a supramolecular complex of water-soluble polythiophene and ATP, Analyst 138 (2013) 1649-1652. [17] Q.L. Xu, K.M. Lee, F. Wang, J. Yoon, Visual detection of copper ions based on azide- and alkyne-functionalized polydiacetylene vesicles, J. Mater. Chem. 21 (2011) 15214-15217. 13
[18] C.-H. Lu, Y.-W. Wang, S.-L. Ye, G.-N. Chen, H.-H. Yang, Ultrasensitive detection of Cu2+ with the naked eye and application in immunoassays, NPG Asia Mater. 4 (2012) 1884-4057. [19] X.R He, H.B. Liu, Y.L. Li, S. Wang, Y.J. Li, N. Wang, J.C. Xiao, X.H. Xu, D.B. Zhu, Gold nanoparticle-based fluorometric and colorimetric sensing of copper(II) ions, Adv. Mater. 17 (2005) 2811-2815. [20] J.H. Huang, Q.B. Zheng, J.-K. Kim, Z.G. Li, A molecular beacon and graphene oxide-based fluorescent biosensor for Cu2+ detection, Biosens. Bioelectron. 43 (2013) 379-383. [21] J.W. Liu, Y, Lu, A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity, J. Am. Chem. Soc. 129 (2007) 9838-9839. [22] H.X. Qin, J.T. Ren, J.H. Wang, E.K. Wang, G-quadruplex facilitated turn-off fluorescent chemosensor for selective detection of cupric ion, Chem. Commun. 46 (2010) 7385-7387. [23] A.W. Zhu, Q. Qu, X.L. Shao, B. Kong, Y. Tian, Carbon-dot-based dual-emission nanohybrid produces a ratiometric fluorescent sensor for in vivo imaging of cellular copper ions, Angew. Chem. Int. Ed. 51 (2012) 7185-7189. [24] H.C. Kolb, M.G. Finn, K.B. Sharpless, Click chemistry: Diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021. [25] M. Meldal, C.W. Tornøe, Cu-catalyzed azide-alkyne cycloaddition, Chem. Rev. 108 (2008) 2952-3015. 14
[26] F. Amblard, J.H. Cho, R.F. Schinazi, The Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide and oligonucleotide chemistry, Chem. Rev. 109 (2009) 4207-4220. [27] T. Efthymiou, W. Gong, J.-P. Desaulniers, Chemical architecture and applications of nucleic acid derivatives containing 1, 2, 3-triazole functionalities synthesized via click chemistry, Molecules 17 (2012) 12665-12703. [28] Y. Zhou, S.X. Wang, K. Zhang, X.Y. Jiang, Visual detection of copper (II) by azide-and alkyne-functionalized gold nanoparticles using click chemistry, Angew. Chem. Int. Ed. 47 (2008) 7454-7456. [29] C. Hua, W.H. Zhang, S.R.M. De Almeida, S. Ciampi, D. Gloria, G.Z. Liu, J.B. Harper, J.J. Gooding, A novel route to copper(II) detection using ‘click’ chemistry-induced aggregation of gold nanoparticles, Analyst 137 (2012) 82-86. [30] S. Park, H.-J. Kim, Highly selective and sensitive fluorescence turn-on probe for a catalytic amount of Cu(I) ions in water through the click reaction, Tetrahedron Lett. 53 (2012) 4473-4475. [31] Z.Y. Lin, S. Gao, J. Lin, W.L. Lin, S.Y. Qiu, L.H. Guo, B. Qiu, G.N. Chen, Visual detection of copper(II) based on the aggregation of gold nano-particles via click chemistry, Anal. Methods-UK 4 (2012) 612-615. [32] A. Lee, J. Chin, O.K. Park, H. Chung, J.W. Kim, S.-Y. Yoon, K. Park, A novel near-infrared fluorescence chemosensor for copper ion detection using click ligation and energy transfer, Chem. Commun. 49 (2013) 5969-5971. [33] J. Su, J. Xu, Y. Chen, Y. Xiang, R. Yuan, Y.Q. Chai, Sensitive detection of 15
copper(II) by a commercial glucometer using click chemistry, Biosens. Bioelectron. 45 (2013) 219-222. [34] T. Sanji, M. Nakamura, M. Tanaka, Fluorescence ‘turn-on’ detection of Cu2+ ions with aggregation-induced emission-active tetraphenylethene based on click chemistry, Tetrahedron Lett. 52 (2011) 3283-3286. [35] X.Y. Xu, W.L. Daniel, W. Wei, C.A. Mirkin, Colorimetric Cu2+ detection using DNA-modified gold-nanoparticle aggregates as probes and click chemistry, Small 6 (2010) 623-626. [36] Q.P. Shen, W.H. Li, S.Y. Tang, Y.F. Hu, Z. Nie, Y. Huang, S.Z. Yao, A simple ‘‘clickable’’ biosensor for colorimetric detection of copper(II) ions based on unmodified gold nanoparticles, Biosens. Bioelectron. 41 (2013) 663-668. [37] L.F. Zhou, Q.P. Shen, P. Zhao, B.B. Xiang, Z. Nie, Y. Huang, S.Z. Yao, Fluorescent detection of copper(II) based on DNA-templated click chemistry and graphene oxide, Methods 64 (2013) 299-304. [38] Q.P. Shen, S.Y. Tang, W.H. Li, Z. Nie, Z.L. Liu, Y. Huang, S.Z. Yao, A novel DNA-templated click chemistry strategy for fluorescent detection of copper(II) ions, Chem. Commun. 48 (2012) 281-283. [39] Q.P. Shen, L.F. Zhou, Y.J. Yuan, Y. Huang, B.B. Xiang, C.Y. Chen, Z. Nie, S.Z.Yao, Intra-molecular G-quadruplex structure generated by DNA-templated click chemistry: “Turn-on” fluorescent probe for copper ions, Biosens. Bioelectron. 55 (2014) 187-194. [40] A. H. El-Sagheer, T. Brown, Click chemistry with DNA, Chem. Soc. Rev. 39 16
(2010) 1388-1405. [41] H.B. Sun, X.H. Peng, Template-directed fluorogenic oligonucleotide ligation using “click” chemistry: Detection of single nucleotide polymorphism in the human p53 tumor suppressor gene, Bioconjugate Chem. 24 (2013) 1226-1234. [42] R. Kumar, A. El-Sagheer, J. Tumpane, P. Lincoln, L.M. Wilhelmsson, T. Brown, Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry, J. Am. Chem. Soc. 129 (2007) 6859-6864. [43] D.S. del Amo, W. Wang, H. Jiang, C. Besanceney, A.C. Yan, M. Levy, Y. Liu, F.L. Marlow, P. Wu, Biocompatible copper (I) catalysts for in vivo imaging of glycans, J. Am. Chem. Soc. 132 (2010) 16893-16899. [44] A.H. El-Sagheer, T. Brown, Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility, Chem. Sci. 5 (2014) 253-259. [45] S. Tyagi, F.R. Kramer, Molecular beacons: Probes that fluoresce upon hybridization, Nat. Biotechnol. 14 (1996) 303-308. [46] K.M. Wang, Z.W. Tang, C.Y.J. Yang, Y.M. Kim, X.H. Fang, W. Li, Y.R. Wu, C.D. Medley, Z.H. Cao, J. Li, P. Colon, H. Lin, W.H. Tan, Molecular engineering of DNA: Molecular beacons, Angew. Chem. Int. Ed. 48 (2009) 856-870. [47] Z.W. Tang, P. Liu, C.B. Ma, X.H. Yang, K.M. Wang, W.H. Tan, X.Y. Lv, Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase, 17
Anal. Chem. 83 (2011) 2505-2510. [48] Z.W. Tang, K.M. Wang, W.H. Tan, C.B. Ma, J. Li, L.F. Liu, Q.P. Guo X.X. Meng, Real-time investigation of nucleic acids phosphorylation process using molecular beacons, Nucleic Acids Res. 33 (2005) e97. [49] F. Chen, Y.X. Zhao, L. Qi, C.H. Fan, One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification Biosens. Bioelectron. 47 (2013) 218-224. [50] Y. Cheng, J.P. Lei, Y.L. Chen, H.X. Ju, Highly selective detection of
microRNA based on distance-dependent electrochemiluminescence resonance energy transfer between CdTe nanocrystals and Au nanoclusters, Biosens. Bioelectron. 51 (2014) 431-436. [51] A.A. Goulko, F. Li, X.C. Le, Bioanalytical applications of aptamer and molecular-beacon probes in fluorescence-affinity assays, Trend Anal. Chem. 28 (2009) 878-892.
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Figure captions Scheme 1. Schematic representation of the copper(II) sensing strategy.
Table 1 The oligonucleotides employed in the current investigation.
Fig. 1 Melting temperature determination of Oligo-MB in 20 mM Tris-HCl (pH 7.4). The solution was equilibrated for 5 min prior to the fluorescence measurement. Data points were taken at 3 ºC intervals. Fluorescence intensity changes were recorded at 514 nm.
Fig. 2 a) Oligo-MB emission spectral changes with reaction time; b) Changes in fluorescence intensity with reaction time with (red) and without (black) the addition of Cu2+. The concentrations of ascorbic acid and Cu2+ were 500 M and 50 nM, respectively. Buffer: 20 mM Tris-HCl (pH 7.4). The emission intensity was recorded at 514 nm at 25 ºC.
Fig. 3 a) Fluorescence intensity changes with reaction time at different reaction temperatures. b) Enhancement of fluorescence intensity after 60 min of reaction at different temperatures. The concentrations of ascorbic acid and Cu2+ were 500 M 19
and 50 nM, respectively. Buffer: 20 mM Tris-HCl (pH 7.4). Emission intensity was recorded at 514 nm.
Fig. 4 Effect of the concentration of ascorbic acid on the performance of the assay. All the solutions were incubated with 50 nM Cu2+ at 25 ºC for 120 min in 20 mM Tris-HCl (pH 7.4). The experiments were repeated three times and the average intensity values were given.
Fig. 5 Changes in emission intensity with Cu2+ concentration (0, 2 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM). The solutions were incubated at 25 ºC for 120 min in 20 mM Tris-HCl (pH 7.4). The concentration of ascorbic acid was 500 M. The experiments were repeated three times and the average intensity values were given.
Fig. 6 Selectivity of the assay. The concentration of Cu2+ was 50 nM and the concentrations of the other metal ions (Ba2+, Mg2+, Ni2+, Co2+, Fe2+, Cd2+, Ca2+, Zn2+, Mn2+, Hg2+, Pb2+, Li+, Na+, K+, Ag+) were 50 M each. The experiments were repeated three times and the average intensity values were given. *Ag+ formed precipitate with Cl- which was removed by centrifugation prior to analysis. If the precipitate was not removed, it caused slight interference with the assay.
Table 2 Copper(II) recovery in lake water samples. 20
Scheme 1.
Table 1 Oligonucleotide sequence (5' 3') Oligo-A
alkyne-GAA ACT GAT AG
Oligo-B
CTA AAT TCC AA-azide
Oligo-MB
FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-Dabcyl
Oligo-A-B
CTA AAT TCC AA-ligated-G AAA CTG ATA G
21
Fig. 1
Fig. 2 22
Fig. 3
23
Fig. 4
Fig. 5
Fig. 6
24
Table 2 Found
Recovery
RSDa
(nM)
(nM)
(%)
(%, n=3)
1
20.0
20.6 ± 0.6
103.0
2.9
2
40.0
39.5 ± 0.9
98.8
2.3
Samples Added
a
RSD: relative standard deviation.
Highlights z
A copper(II) detection method with good sensitivity and selectivity
z
The click reaction could be monitored in a real-time fashion
z
A molecular beacon was used as the template
z
A fluorescence turn-on strategy reduces the possibility of false positive signals
25