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A turn-on fluorescent aptasensor for adenosine detection based on split aptamers and graphene oxide† Yunfeng Bai,ab Feng Feng,*ab Lu Zhao,b Zezhong Chen,b Haiyan Wangb and Yali Duana
DOI: 10.1039/c4an00084f www.rsc.org/analyst
A simple, sensitive and selective turn-on fluorescent aptasensor for adenosine detection was developed based on target-induced split aptamer fragment conjunction and different interactions of graphene oxide and the two states of the designed aptamer sequences.
Adenosine is an important endogenous modulator, present in all cells, which plays a role in the regulation of physiological activity in various tissues and organs.1 In the central nervous system, adenosine can regulate cerebral blood ow and modulate neurotransmission.2 Adenosine also protects against neuronal damage caused by oxidative stress and has been studied for possible protective effects in hypoglycemia, hypoxia, and ischemia.1 Furthermore, some evidence indicates that it has some important functions in the immune system.3 Therefore, the simple and sensitive detection of adenosine has considerable practical value. Aptamers are in vitro selected functional oligonucleotides that can bind specically to target molecules.4 In principle, aptamers with high affinity can be selected for virtually any given target via a process termed systematic evolution of ligands by exponential enrichment (SELEX).4a,4b,5 As increasingly important biosensing elements, aptamers have well exhibited their competitive advantages over other natural or articial receptors (e.g., antibodies or molecularly imprinted polymers), such as synthesis convenience, ease of chemical modication, chemical stability, and exibility in biosensor design.6 The adenosine binding DNA aptamer was rst isolated and characterized by Szostak and co-workers in 1995.7 So far, several methods for the quantitative determination of adenosine based on aptamers have been reported involving various
a
School of Chemistry and Materials Science, Shanxi Normal University, Linfen 041004, P. R. China. E-mail: [email protected]; Fax: +86-352-6100028; Tel: +86-3527158662
b
College of Chemistry and Chemical Engineering, Shanxi Datong University, Datong 037009, P. R. China † Electronic supplementary 10.1039/c4an00084f
information
(ESI)
This journal is © The Royal Society of Chemistry 2014
available.
See
DOI:
signal-transduction approaches such as uorescence,8 electrochemistry,9 electrochemiluminescence (ECL),10 colorimetry,11 and surface-enhanced Raman scattering (SERS).12 Actually, aptamers are more inclined to form aptamer/target complexes but not to form aptamer/ssDNA duplexes.7 Therefore, most of these strategies are based on target-induced switching between an aptamer/ssDNA duplex and an aptamer/ target complex. Split aptamers are composed of two nucleic acid strands that assemble selectively in the presence of a small molecule or protein target.7,13 Recently, the construction of aptasensors based on the self-assembly of split aptamers was introduced as a general platform for small molecules.14 According to this method, the aptamer is divided into two subunits that do not interact with one another in the absence of an analyte. However, in the presence of the respective analyte, a tricomponent supramolecular aptamer complex is generated. Graphene oxide (GO) chemically exfoliated from oxidized graphite is considered as a promising material for biological applications owing to its excellent aqueous processability, amphiphilicity, surface functionalizability, and uorescence quenching ability.15 Based on these properties, several GObased sensors have been developed for the detection of DNA,16 proteins,17 small molecules,18 and harmful metal ions19 by using dye-labeled complementary oligonucleotides or aptamers as recognition elements. However, despite its superb properties, the GO based platform has rarely been applied to detect the small molecules based on split aptamers. Here, for the rst time, we fabricate a turn-on uorescent aptasensor for adenosine by taking advantage of the high binding specicity of split aptamers and unique interactions of graphene oxide and the two states of the designed aptamer sequences. Compared with the previous adenosine assay methods, the proposed strategy is simple, cost effective and selective, which might become a new method of choice for sensitive small molecule detection.
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Scheme 1 Schematic illustration of the design strategy involved in the fluorescent aptasensor for adenosine based on the GO platform.
The mechanism of the proposed uorescent aptasensor for adenosine based on the GO platform is shown in Scheme 1. The adenosine aptamer is cut into two exible ssDNA fragments (ABA1-FAM: 50 -FAM-ACC TGG GGG AGT AT-30 and ABA2: 50 -TGC GGA GGA AGG T-30 ) according to the literature.13a,14c FAMlabeled ABA1 and GO readily form stable ABA1-FAM/GO complexes in aqueous solution by means of p–p stacking interactions between nucleotide bases and honeycomb lattices of GO, which results in complete uorescence quenching by the efficient long range energy transfer from the dye to GO.18c In the absence of adenosine, when ABA2 is added, ABA1-FAM and ABA2 could not form stable intermolecular duplexes, and no apparent uorescence enhancement is observed. However, in the presence of adenosine, the two fragments assemble into an intact tertiary structure. Consequently, the weak binding between the ABA1-FAM/adenosine/ABA2 complex and GO surface makes the dyes far away from the GO surface and leads to uorescence restoration. The concentration of adenosine could be correlated quantitatively to the increase of uorescence intensity of the system. To demonstrate the feasibility of the proposed aptasensor, the uorescence intensity of the ABA1-FAM under different conditions was measured. As shown in Fig. 1A, ABA1-FAM shows a strong uorescence emission owing to the presence of the uorescein-based dye (Fig. 1A, curve a). However, in the presence of GO, up to 98% quenching of the uorescence emission is observed (Fig. 1A, curve d), indicating the strong adsorption of ABA1-FAM on GO and the high uorescence quenching efficiency of GO. The uorescence intensity is rarely
Fluorescence emission spectra (A) and fluorescence anisotropy (B) of ABA1-FAM under different conditions: (a) ABA1-FAM; (b) ABA1FAM + ABA2; (c) ABA1-FAM + ABA2 + adenosine; (d) ABA1-FAM + GO; (e) ABA1-FAM + ABA2 + GO; (f) ABA1-FAM + ABA2 + adenosine + GO. Concentrations of ABA1-FAM, ABA2, adenosine and GO are 50 nM, 50 nM, 1 mM and 18 mg ml1, respectively. The excitation wavelength is 480 nm. Fig. 1
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affected by the addition of ABA2 (Fig. 1A, curve e), suggesting that ABA1-FAM and ABA2 could not form stable intermolecular duplexes. Upon the addition of ABA2 and adenosine, adenosine reassembles the two pieces of ssDNA into the intact aptamer tertiary structure which releases from the GO surface. Consequently, the FAM molecules are far away from the GO surface and the energy transfer efficiency decreases, then the quenched uorescence recovers remarkably (Fig. 1A, curve f). However, the uorescence intensity of free ABA1-FAM is scarcely inuenced by the addition of ABA2 and adenosine (Fig. 1A, curve b and c). These results demonstrate the strong interaction between the split aptamer and GO and the competitive binding of adenosine and GO with the split aptamer. The uorescence anisotropy (FA) measurement provides the rotation information of a uorophore in its microenvironment, which is closely related to the size, shape, and interaction process of the uorophore.20 To further understand the interaction between ABA1-FAM, ABA2, adenosine and GO in solution, uorescence anisotropy of ABA1-FAM under different conditions was measured. As shown in Fig. 1B, the original FA value of ABA1-FAM is 0.044. However, the addition of GO causes a signicant FA value increase of ABA1-FAM to 0.45, indicating that binding of GO to ABA1-FAM leads to a larger mass complex, which hinders the rotation diffusion rate of the labeled FAM. Aer the addition of ABA2, the FA value remains high, and a bit bigger than that of the ABA1-FAM/GO complex, probably due to the adsorption of ABA2 onto the GO surface. While aer the addition of ABA2 and adenosine, a signicant anisotropy reduction is observed from 0.45 to 0.093. This illustrates that adenosine reassembles the two pieces of ssDNA into the intact aptamer tertiary structure and then detaches from the GO, making the dyes far away from the GO surface. Addition of ABA2 and adenosine to the ABA1-FAM solution produces little measurable change in anisotropy. This illustrates that the molecular mass of adenosine is relatively too small to hinder the rotational diffusion rate of the ABA1-FAM. The abovementioned experimental results clearly demonstrate the feasibility of the proposed strategy for sensitive adenosine detection. In order to achieve the best assay performance, the effects of GO concentration on the uorescence intensity of ABA1-FAM/ ABA2 in the absence and presence of adenosine were
Fig. 2 (A) Fluorescence intensity of ABA1-FAM/ABA2 in the absence (a) and presence (b) of adenosine upon addition of different concentrations of GO. (B) The fluorescence enhancement of ABA1-FAM/ABA2 by adenosine as a function of GO concentration. Concentrations of ABA1-FAM, ABA2, and adenosine are 50 nM, 50 nM, and 1 mM, respectively. The excitation and emission wavelengths are 480 and 519 nm.
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investigated. As shown in Fig. 2A, the uorescence intensity of ABA1-FAM/ABA2 decreases sharply as the concentration of GO increases either in the absence or presence of adenosine. The highest uorescence enhancement ratio (F/F0, where F0 and F are the uorescence intensities of ABA1-FAM/ABA2 with different concentrations of GO in the absence and presence of adenosine, respectively) is obtained at [GO] ¼ 18 mg ml1 (Fig. 2B). This clearly demonstrates that a moderate amount of GO could reduce the background signal and improve the analytical sensitivity. On the basis of these results, a concentration of GO at 18 mg ml1 is chosen for the subsequent experiments. To investigate the sensitivity of the sensing platform, the uorescence emission spectra of ABA1-FAM/ABA2/GO solution spiked with various concentrations of adenosine are shown in Fig. 3A. The uorescence intensity of the system enhances dramatically with the increasing concentration of adenosine. In Fig. 3B, the uorescence enhancement ratio, F/F0, at lex/lem ¼ 480/519 nm, is plotted as a function of the adenosine concentration, where F and F0 are the system uorescence intensities in the presence and absence of adenosine, respectively. A linear relationship between the uorescence enhancement ratio of the solution and the concentration of adenosine is observed in the range of 0–1400 mM (R2 ¼ 0.9961) with the detection limit of 6 mM based on the 3s/S calculation (s is the standard deviation for the blank solution and S is the slope of the calibration curve), which is superior or comparable to the aptamer based methods.8b,8d,14c,21 The results indicate that this uorescent aptasensor possesses the potential of sensitive assay of adenosine in aqueous solution. To demonstrate the selectivity of our design for adenosine assay, the uorescence responses of the aptasensor to adenosine analogues including thymidine, cytidine, uridine, and mixed sample were tested. Fig. 4 exhibits different uorescence enhancement signals of the proposed sensing system aer the addition of 1 mM adenosine, thymidine, cytidine, uridine or mixed sample under the same experimental conditions. Incubations of ABA1-FAM/ABA2/GO with thymidine, cytidine or uridine do not produce any signicant
Fig. 3 (A) Fluorescence emission spectra of ABA1-FAM/ABA2/GO upon addition of different concentrations of adenosine. The arrow indicates the signal changes with increasing adenosine concentrations (0, 20, 40, 80, 120, 160, 200, 300, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 mM). (B) Calibration curve for adenosine detection. The inset shows the linear relationship between the fluorescence enhancement ratio and the adenosine concentration over the range of 0–1400 mM. The fluorescence intensity was recorded at 519 nm with an excitation wavelength of 480 nm. Error bars represent the standard deviations for three replicates.
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Fluorescence enhancement ratio of the proposed aptasensor in the presence of 1 mM adenosine, 1 mM of its analogues and mixed sample. The fluorescence intensity was recorded at 519 nm with an excitation wavelength of 480 nm. Error bars represent the standard deviations for three replicates.
Fig. 4
change in the uorescence intensity response as compared to the case of adenosine, which implies that the engineered split aptamer retains its high selectivity toward adenosine molecules and is able to discriminate adenosine from its analogues. The application of the proposed method was evaluated for the determination of adenosine in serum samples. The serum 10-fold diluted with tris–HCl buffer (20 mM, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, pH 7.4) was used to prepare the adenosine solution and other experimental conditions were the same as described in the sensitivity assay. As shown in Table 1, the recovery of the added adenosine is in the range of 98–106%, indicating that the proposed aptasensor could still work in the complex serum matrix and it might be promising for the determination of adenosine in clinical examination. In conclusion, a simple mix-and-detect turn-on uorescent aptasensor for adenosine has been developed. The proposed sensing system combines the selective recognition property of split aptamers with different adsorption affinities between GO and DNA structures. Furthermore, this strategy possesses some prominent advantages. Firstly, the assay can be operated by mixing the split aptamer, GO, and the target molecule, which offers a convenient protocol for simple and cost-effective adenosine detection. Secondly, the background signal is signicantly reduced owing to the high energy transfer efficiency between the GO and the uorophore. Thirdly, given rapid advances in split aptamer engineering technologies,22 it appears that the split aptamer–GO system might provide a new universal platform for selective detection of a wide range of analytes by using specic split aptamers. Table 1 Determination of adenosine in serum samples using the proposed method
Samples
Added (mM)
Found, mean SD (mM, n ¼ 3)
1 2 3 4
300 500 1000 1400
318 514 1045 1368
15 26 48 65
Recovery (%) 106 103 105 98
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Acknowledgements
Published on 10 February 2014. Downloaded by University of Illinois at Chicago on 27/10/2014 02:49:35.
This work was supported by the National Natural Science Foundation of China (21175085 and 21375083) and the Natural Science Foundation of Shanxi Province (2009011015-1).
Notes and references 1 S. Latini and F. Pedata, J. Neurochem., 2001, 79, 463. 2 J. M. Brundege and T. V. Dunwiddie, Adv. Pharmacol., 1997, 39, 353. 3 A. Ohta and M. Sitkovsky, Nature, 2001, 414, 916. 4 (a) A. D. Ellington and J. W. Szostak, Nature, 1990, 346, 818; (b) C. Tuerk and L. Gold, Science, 1990, 249, 505; (c) D. L. Robertson and G. F. Joyce, Nature, 1990, 344, 467. 5 A. D. Ellington and J. W. Szostak, Nature, 1992, 355, 850. 6 (a) D. Li, S. Song and C. Fan, Acc. Chem. Res., 2010, 43, 631; (b) J. Liu, Z. Cao and Y. Lu, Chem. Rev., 2009, 109, 1948; (c) M. Mascini, I. Palchetti and S. Tombelli, Angew. Chem., Int. Ed., 2012, 51, 1316; (d) M. Famulok and G. Mayer, Acc. Chem. Res., 2011, 44, 1349; (e) Z. Wu, L. Tang, X. Zhang, J. Jiang and W. Tan, ACS Nano, 2011, 5, 7696. 7 D. E. Huizenga and J. W. Szostak, Biochemistry, 1995, 34, 656. 8 (a) Y. Xiang, A. J. Tong and Y. Lu, J. Am. Chem. Soc., 2009, 131, 15352; (b) B. Fu, J. Cao, W. Jiang and L. Wang, Biosens. Bioelectron., 2013, 44, 52; (c) L. Li, P. Ge, P. R. Selvin and Y. Lu, Anal. Chem., 2012, 84, 7852; (d) P. J. J. Huang and J. W. Liu, Anal. Chem., 2010, 82, 4020. 9 (a) F. Shahdost-fard, A. Salimi and S. Khezrian, Biosens. Bioelectron., 2014, 53, 355; (b) S. S. Zhang, J. P. Xia and X. M. Li, Anal. Chem., 2008, 80, 8382; (c) Z. Wu, M. Guo, S. Zhang, C. Chen, J. Jiang, G. Shen and R. Yu, Anal. Chem., 2007, 79, 2933. 10 (a) X. Zhu, Y. S. Zhang, W. Q. Yang, Q. D. Liu, Z. Y. Lin, B. Qiu and G. N. Chen, Anal. Chim. Acta, 2011, 684, 121; (b) X. Y. Wang, P. Dong, P. G. He and Y. Z. Fang, Anal. Chim. Acta, 2010, 658, 128. 11 Z. F. Liu, J. Ge and X. S. Zhao, Chem. Commun., 2011, 47, 4956. 12 J. W. Chen, X. P. Liu, K. J. Feng, Y. Liang, J. H. Jiang, G. L. Shen and R. Q. Yu, Biosens. Bioelectron., 2008, 24, 66. 13 (a) J. Zhang, L. H. Wang, D. Pan, S. P. Song, F. Y. C. Boey, H. Zhang and C. H. Fan, Small, 2008, 4, 1196; (b) R. Yamamoto-Fujita and P. K. R. Kumar, Anal. Chem., 2005, 77, 5460. 14 (a) X. L. Zuo, Y. Xiao and K. W. Plaxco, J. Am. Chem. Soc., 2009, 131, 6944; (b) R. Freeman, E. Sharon, R. Tel-Vered and I. Willner, J. Am. Chem. Soc., 2009, 131, 5028; (c) F. Li, J. Zhang, X. Cao, L. Wang, D. Li, S. Song, B. Ye and C. Fan,
1846 | Analyst, 2014, 139, 1843–1846
15
16
17
18
19
20 21 22
Analyst, 2009, 134, 1355; (d) X. He, Z. Li, X. Jia, K. Wang and J. Yin, Talanta, 2013, 111, 105; (e) F. Jin, Y. Lian, J. Li, J. Zheng, Y. Hu, J. Liu, J. Huang and R. Yang, Anal. Chim. Acta, 2013, 799, 44; (f) S. Cheng, B. Zheng, M. Wang, M. H.-W. Lam and X. Ge, Talanta, 2013, 115, 506; (g) J. Chen and L. Zeng, Biosens. Bioelectron., 2013, 42, 93; (h) A. K. Sharma, A. D. Kent and J. M. Heemstra, Anal. Chem., 2012, 84, 6104; (i) X. Xu, J. Zhang, F. Yang and X. Yang, Chem. Commun., 2011, 47, 9435; (j) J. Chen, Z. Fang, P. Lie and L. Zeng, Anal. Chem., 2012, 84, 6321; (k) J. Lu, M. Yan, L. Ge, S. Ge, S. Wang, J. Yan and J. Yu, Biosens. Bioelectron., 2013, 47, 271; (l) J. Liu, Y. Liu, X. Yang, K. Wang, Q. Wang, H. Shi and L. Li, Anal. Chem., 2013, 85, 11121. (a) D. Chen, H. Feng and J. Li, Chem. Rev., 2012, 112, 6027; (b) C. Chung, Y.-K. Kim, D. Shin, S.-R. Ryoo, B. H. Hong and D.-H. Min, Acc. Chem. Res., 2013, 46, 2211. (a) C. H. Lu, H. H. Yang, C. L. Zhu, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2009, 48, 4785; (b) S. He, B. Song, D. Li, C. Zhu, W. Qi, Y. Wen, L. Wang, S. Song, H. Fang and C. Fan, Adv. Funct. Mater., 2010, 20, 453; (c) X. Liu, R. Aizen, R. Freeman, O. Yehezkeli and I. Willner, ACS Nano, 2012, 6, 3553. (a) H. Chang, L. Tang, Y. Wang, J. Jiang and J. Li, Anal. Chem., 2010, 82, 2341; (b) L. Wang, J. Zhu, L. Han, L. Jin, C. Zhu, E. Wang and S. Dong, ACS Nano, 2012, 6, 6659; (c) M. Zhang, B. Yin, W. Tan and B. Ye, Biosens. Bioelectron., 2011, 26, 3260. (a) C. H. Lu, J. A. Li, M. H. Lin, Y. W. Wang, H. H. Yang, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2010, 49, 8454; (b) Y. Wang, Z. Li, D. Hu, C.-T. Lin, J. Li and Y. Lin, J. Am. Chem. Soc., 2010, 132, 9274; (c) Y. Wang, Z. Li, T. J. Weber, D. Hu, C.-T. Lin, J. Li and Y. Lin, Anal. Chem., 2013, 85, 6775; (d) P. Zhang, Y. Wang, F. Leng, Z. H. Xiong and C. Z. Huang, Talanta, 2013, 112, 117. (a) Y. Wen, F. Xing, S. He, S. Song, L. Wang, Y. Long, D. Li and C. Fan, Chem. Commun., 2010, 46, 2596; (b) X. Zhao, R. Kong, X. Zhang, H. Meng, W. Liu, W. Tan, G. Shen and R. Yu, Anal. Chem., 2011, 83, 5062; (c) W. Y. Xie, W. T. Huang, N. B. Li and H. Q. Luo, Chem. Commun., 2012, 48, 82; (d) M. Li, X. Zhou, S. Guo and N. Wu, Biosens. Bioelectron., 2013, 43, 69; (e) Y. Yu, Y. Liu, S. J. Zhen and C. Z. Huang, Chem. Commun., 2013, 49, 1942. J. Liu, C. Wang, Y. Jiang, Y. Hu, J. Li, S. Yang, Y. Li, R. Yang, W. Tan and C. Z. Huang, Anal. Chem., 2013, 85, 1424. (a) D. Liao, H. Jiao, B. Wang, Q. Lin and C. Yu, Analyst, 2012, 137, 978; (b) W. C. Xu and Y. Lu, Anal. Chem., 2010, 82, 574. A. D. Kent, N. G. Spiropulos and J. M. Heemstra, Anal. Chem., 2013, 85, 9916.
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Cite this: Analyst, 2014, 139, 1843
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A turn-on fluorescent aptasensor for adenosine detection based on split aptamers and graphene oxide† Yunfeng Bai,ab Feng Feng,*ab Lu Zhao,b Zezhong Chen,b Haiyan Wangb and Yali Duana
DOI: 10.1039/c4an00084f www.rsc.org/analyst
A simple, sensitive and selective turn-on fluorescent aptasensor for adenosine detection was developed based on target-induced split aptamer fragment conjunction and different interactions of graphene oxide and the two states of the designed aptamer sequences.
Adenosine is an important endogenous modulator, present in all cells, which plays a role in the regulation of physiological activity in various tissues and organs.1 In the central nervous system, adenosine can regulate cerebral blood ow and modulate neurotransmission.2 Adenosine also protects against neuronal damage caused by oxidative stress and has been studied for possible protective effects in hypoglycemia, hypoxia, and ischemia.1 Furthermore, some evidence indicates that it has some important functions in the immune system.3 Therefore, the simple and sensitive detection of adenosine has considerable practical value. Aptamers are in vitro selected functional oligonucleotides that can bind specically to target molecules.4 In principle, aptamers with high affinity can be selected for virtually any given target via a process termed systematic evolution of ligands by exponential enrichment (SELEX).4a,4b,5 As increasingly important biosensing elements, aptamers have well exhibited their competitive advantages over other natural or articial receptors (e.g., antibodies or molecularly imprinted polymers), such as synthesis convenience, ease of chemical modication, chemical stability, and exibility in biosensor design.6 The adenosine binding DNA aptamer was rst isolated and characterized by Szostak and co-workers in 1995.7 So far, several methods for the quantitative determination of adenosine based on aptamers have been reported involving various
a
School of Chemistry and Materials Science, Shanxi Normal University, Linfen 041004, P. R. China. E-mail: [email protected]; Fax: +86-352-6100028; Tel: +86-3527158662
b
College of Chemistry and Chemical Engineering, Shanxi Datong University, Datong 037009, P. R. China † Electronic supplementary 10.1039/c4an00084f
information
(ESI)
This journal is © The Royal Society of Chemistry 2014
available.
See
DOI:
signal-transduction approaches such as uorescence,8 electrochemistry,9 electrochemiluminescence (ECL),10 colorimetry,11 and surface-enhanced Raman scattering (SERS).12 Actually, aptamers are more inclined to form aptamer/target complexes but not to form aptamer/ssDNA duplexes.7 Therefore, most of these strategies are based on target-induced switching between an aptamer/ssDNA duplex and an aptamer/ target complex. Split aptamers are composed of two nucleic acid strands that assemble selectively in the presence of a small molecule or protein target.7,13 Recently, the construction of aptasensors based on the self-assembly of split aptamers was introduced as a general platform for small molecules.14 According to this method, the aptamer is divided into two subunits that do not interact with one another in the absence of an analyte. However, in the presence of the respective analyte, a tricomponent supramolecular aptamer complex is generated. Graphene oxide (GO) chemically exfoliated from oxidized graphite is considered as a promising material for biological applications owing to its excellent aqueous processability, amphiphilicity, surface functionalizability, and uorescence quenching ability.15 Based on these properties, several GObased sensors have been developed for the detection of DNA,16 proteins,17 small molecules,18 and harmful metal ions19 by using dye-labeled complementary oligonucleotides or aptamers as recognition elements. However, despite its superb properties, the GO based platform has rarely been applied to detect the small molecules based on split aptamers. Here, for the rst time, we fabricate a turn-on uorescent aptasensor for adenosine by taking advantage of the high binding specicity of split aptamers and unique interactions of graphene oxide and the two states of the designed aptamer sequences. Compared with the previous adenosine assay methods, the proposed strategy is simple, cost effective and selective, which might become a new method of choice for sensitive small molecule detection.
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Scheme 1 Schematic illustration of the design strategy involved in the fluorescent aptasensor for adenosine based on the GO platform.
The mechanism of the proposed uorescent aptasensor for adenosine based on the GO platform is shown in Scheme 1. The adenosine aptamer is cut into two exible ssDNA fragments (ABA1-FAM: 50 -FAM-ACC TGG GGG AGT AT-30 and ABA2: 50 -TGC GGA GGA AGG T-30 ) according to the literature.13a,14c FAMlabeled ABA1 and GO readily form stable ABA1-FAM/GO complexes in aqueous solution by means of p–p stacking interactions between nucleotide bases and honeycomb lattices of GO, which results in complete uorescence quenching by the efficient long range energy transfer from the dye to GO.18c In the absence of adenosine, when ABA2 is added, ABA1-FAM and ABA2 could not form stable intermolecular duplexes, and no apparent uorescence enhancement is observed. However, in the presence of adenosine, the two fragments assemble into an intact tertiary structure. Consequently, the weak binding between the ABA1-FAM/adenosine/ABA2 complex and GO surface makes the dyes far away from the GO surface and leads to uorescence restoration. The concentration of adenosine could be correlated quantitatively to the increase of uorescence intensity of the system. To demonstrate the feasibility of the proposed aptasensor, the uorescence intensity of the ABA1-FAM under different conditions was measured. As shown in Fig. 1A, ABA1-FAM shows a strong uorescence emission owing to the presence of the uorescein-based dye (Fig. 1A, curve a). However, in the presence of GO, up to 98% quenching of the uorescence emission is observed (Fig. 1A, curve d), indicating the strong adsorption of ABA1-FAM on GO and the high uorescence quenching efficiency of GO. The uorescence intensity is rarely
Fluorescence emission spectra (A) and fluorescence anisotropy (B) of ABA1-FAM under different conditions: (a) ABA1-FAM; (b) ABA1FAM + ABA2; (c) ABA1-FAM + ABA2 + adenosine; (d) ABA1-FAM + GO; (e) ABA1-FAM + ABA2 + GO; (f) ABA1-FAM + ABA2 + adenosine + GO. Concentrations of ABA1-FAM, ABA2, adenosine and GO are 50 nM, 50 nM, 1 mM and 18 mg ml1, respectively. The excitation wavelength is 480 nm. Fig. 1
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affected by the addition of ABA2 (Fig. 1A, curve e), suggesting that ABA1-FAM and ABA2 could not form stable intermolecular duplexes. Upon the addition of ABA2 and adenosine, adenosine reassembles the two pieces of ssDNA into the intact aptamer tertiary structure which releases from the GO surface. Consequently, the FAM molecules are far away from the GO surface and the energy transfer efficiency decreases, then the quenched uorescence recovers remarkably (Fig. 1A, curve f). However, the uorescence intensity of free ABA1-FAM is scarcely inuenced by the addition of ABA2 and adenosine (Fig. 1A, curve b and c). These results demonstrate the strong interaction between the split aptamer and GO and the competitive binding of adenosine and GO with the split aptamer. The uorescence anisotropy (FA) measurement provides the rotation information of a uorophore in its microenvironment, which is closely related to the size, shape, and interaction process of the uorophore.20 To further understand the interaction between ABA1-FAM, ABA2, adenosine and GO in solution, uorescence anisotropy of ABA1-FAM under different conditions was measured. As shown in Fig. 1B, the original FA value of ABA1-FAM is 0.044. However, the addition of GO causes a signicant FA value increase of ABA1-FAM to 0.45, indicating that binding of GO to ABA1-FAM leads to a larger mass complex, which hinders the rotation diffusion rate of the labeled FAM. Aer the addition of ABA2, the FA value remains high, and a bit bigger than that of the ABA1-FAM/GO complex, probably due to the adsorption of ABA2 onto the GO surface. While aer the addition of ABA2 and adenosine, a signicant anisotropy reduction is observed from 0.45 to 0.093. This illustrates that adenosine reassembles the two pieces of ssDNA into the intact aptamer tertiary structure and then detaches from the GO, making the dyes far away from the GO surface. Addition of ABA2 and adenosine to the ABA1-FAM solution produces little measurable change in anisotropy. This illustrates that the molecular mass of adenosine is relatively too small to hinder the rotational diffusion rate of the ABA1-FAM. The abovementioned experimental results clearly demonstrate the feasibility of the proposed strategy for sensitive adenosine detection. In order to achieve the best assay performance, the effects of GO concentration on the uorescence intensity of ABA1-FAM/ ABA2 in the absence and presence of adenosine were
Fig. 2 (A) Fluorescence intensity of ABA1-FAM/ABA2 in the absence (a) and presence (b) of adenosine upon addition of different concentrations of GO. (B) The fluorescence enhancement of ABA1-FAM/ABA2 by adenosine as a function of GO concentration. Concentrations of ABA1-FAM, ABA2, and adenosine are 50 nM, 50 nM, and 1 mM, respectively. The excitation and emission wavelengths are 480 and 519 nm.
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investigated. As shown in Fig. 2A, the uorescence intensity of ABA1-FAM/ABA2 decreases sharply as the concentration of GO increases either in the absence or presence of adenosine. The highest uorescence enhancement ratio (F/F0, where F0 and F are the uorescence intensities of ABA1-FAM/ABA2 with different concentrations of GO in the absence and presence of adenosine, respectively) is obtained at [GO] ¼ 18 mg ml1 (Fig. 2B). This clearly demonstrates that a moderate amount of GO could reduce the background signal and improve the analytical sensitivity. On the basis of these results, a concentration of GO at 18 mg ml1 is chosen for the subsequent experiments. To investigate the sensitivity of the sensing platform, the uorescence emission spectra of ABA1-FAM/ABA2/GO solution spiked with various concentrations of adenosine are shown in Fig. 3A. The uorescence intensity of the system enhances dramatically with the increasing concentration of adenosine. In Fig. 3B, the uorescence enhancement ratio, F/F0, at lex/lem ¼ 480/519 nm, is plotted as a function of the adenosine concentration, where F and F0 are the system uorescence intensities in the presence and absence of adenosine, respectively. A linear relationship between the uorescence enhancement ratio of the solution and the concentration of adenosine is observed in the range of 0–1400 mM (R2 ¼ 0.9961) with the detection limit of 6 mM based on the 3s/S calculation (s is the standard deviation for the blank solution and S is the slope of the calibration curve), which is superior or comparable to the aptamer based methods.8b,8d,14c,21 The results indicate that this uorescent aptasensor possesses the potential of sensitive assay of adenosine in aqueous solution. To demonstrate the selectivity of our design for adenosine assay, the uorescence responses of the aptasensor to adenosine analogues including thymidine, cytidine, uridine, and mixed sample were tested. Fig. 4 exhibits different uorescence enhancement signals of the proposed sensing system aer the addition of 1 mM adenosine, thymidine, cytidine, uridine or mixed sample under the same experimental conditions. Incubations of ABA1-FAM/ABA2/GO with thymidine, cytidine or uridine do not produce any signicant
Fig. 3 (A) Fluorescence emission spectra of ABA1-FAM/ABA2/GO upon addition of different concentrations of adenosine. The arrow indicates the signal changes with increasing adenosine concentrations (0, 20, 40, 80, 120, 160, 200, 300, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 mM). (B) Calibration curve for adenosine detection. The inset shows the linear relationship between the fluorescence enhancement ratio and the adenosine concentration over the range of 0–1400 mM. The fluorescence intensity was recorded at 519 nm with an excitation wavelength of 480 nm. Error bars represent the standard deviations for three replicates.
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Fluorescence enhancement ratio of the proposed aptasensor in the presence of 1 mM adenosine, 1 mM of its analogues and mixed sample. The fluorescence intensity was recorded at 519 nm with an excitation wavelength of 480 nm. Error bars represent the standard deviations for three replicates.
Fig. 4
change in the uorescence intensity response as compared to the case of adenosine, which implies that the engineered split aptamer retains its high selectivity toward adenosine molecules and is able to discriminate adenosine from its analogues. The application of the proposed method was evaluated for the determination of adenosine in serum samples. The serum 10-fold diluted with tris–HCl buffer (20 mM, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, pH 7.4) was used to prepare the adenosine solution and other experimental conditions were the same as described in the sensitivity assay. As shown in Table 1, the recovery of the added adenosine is in the range of 98–106%, indicating that the proposed aptasensor could still work in the complex serum matrix and it might be promising for the determination of adenosine in clinical examination. In conclusion, a simple mix-and-detect turn-on uorescent aptasensor for adenosine has been developed. The proposed sensing system combines the selective recognition property of split aptamers with different adsorption affinities between GO and DNA structures. Furthermore, this strategy possesses some prominent advantages. Firstly, the assay can be operated by mixing the split aptamer, GO, and the target molecule, which offers a convenient protocol for simple and cost-effective adenosine detection. Secondly, the background signal is signicantly reduced owing to the high energy transfer efficiency between the GO and the uorophore. Thirdly, given rapid advances in split aptamer engineering technologies,22 it appears that the split aptamer–GO system might provide a new universal platform for selective detection of a wide range of analytes by using specic split aptamers. Table 1 Determination of adenosine in serum samples using the proposed method
Samples
Added (mM)
Found, mean SD (mM, n ¼ 3)
1 2 3 4
300 500 1000 1400
318 514 1045 1368
15 26 48 65
Recovery (%) 106 103 105 98
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Acknowledgements
Published on 10 February 2014. Downloaded by University of Illinois at Chicago on 27/10/2014 02:49:35.
This work was supported by the National Natural Science Foundation of China (21175085 and 21375083) and the Natural Science Foundation of Shanxi Province (2009011015-1).
Notes and references 1 S. Latini and F. Pedata, J. Neurochem., 2001, 79, 463. 2 J. M. Brundege and T. V. Dunwiddie, Adv. Pharmacol., 1997, 39, 353. 3 A. Ohta and M. Sitkovsky, Nature, 2001, 414, 916. 4 (a) A. D. Ellington and J. W. Szostak, Nature, 1990, 346, 818; (b) C. Tuerk and L. Gold, Science, 1990, 249, 505; (c) D. L. Robertson and G. F. Joyce, Nature, 1990, 344, 467. 5 A. D. Ellington and J. W. Szostak, Nature, 1992, 355, 850. 6 (a) D. Li, S. Song and C. Fan, Acc. Chem. Res., 2010, 43, 631; (b) J. Liu, Z. Cao and Y. Lu, Chem. Rev., 2009, 109, 1948; (c) M. Mascini, I. Palchetti and S. Tombelli, Angew. Chem., Int. Ed., 2012, 51, 1316; (d) M. Famulok and G. Mayer, Acc. Chem. Res., 2011, 44, 1349; (e) Z. Wu, L. Tang, X. Zhang, J. Jiang and W. Tan, ACS Nano, 2011, 5, 7696. 7 D. E. Huizenga and J. W. Szostak, Biochemistry, 1995, 34, 656. 8 (a) Y. Xiang, A. J. Tong and Y. Lu, J. Am. Chem. Soc., 2009, 131, 15352; (b) B. Fu, J. Cao, W. Jiang and L. Wang, Biosens. Bioelectron., 2013, 44, 52; (c) L. Li, P. Ge, P. R. Selvin and Y. Lu, Anal. Chem., 2012, 84, 7852; (d) P. J. J. Huang and J. W. Liu, Anal. Chem., 2010, 82, 4020. 9 (a) F. Shahdost-fard, A. Salimi and S. Khezrian, Biosens. Bioelectron., 2014, 53, 355; (b) S. S. Zhang, J. P. Xia and X. M. Li, Anal. Chem., 2008, 80, 8382; (c) Z. Wu, M. Guo, S. Zhang, C. Chen, J. Jiang, G. Shen and R. Yu, Anal. Chem., 2007, 79, 2933. 10 (a) X. Zhu, Y. S. Zhang, W. Q. Yang, Q. D. Liu, Z. Y. Lin, B. Qiu and G. N. Chen, Anal. Chim. Acta, 2011, 684, 121; (b) X. Y. Wang, P. Dong, P. G. He and Y. Z. Fang, Anal. Chim. Acta, 2010, 658, 128. 11 Z. F. Liu, J. Ge and X. S. Zhao, Chem. Commun., 2011, 47, 4956. 12 J. W. Chen, X. P. Liu, K. J. Feng, Y. Liang, J. H. Jiang, G. L. Shen and R. Q. Yu, Biosens. Bioelectron., 2008, 24, 66. 13 (a) J. Zhang, L. H. Wang, D. Pan, S. P. Song, F. Y. C. Boey, H. Zhang and C. H. Fan, Small, 2008, 4, 1196; (b) R. Yamamoto-Fujita and P. K. R. Kumar, Anal. Chem., 2005, 77, 5460. 14 (a) X. L. Zuo, Y. Xiao and K. W. Plaxco, J. Am. Chem. Soc., 2009, 131, 6944; (b) R. Freeman, E. Sharon, R. Tel-Vered and I. Willner, J. Am. Chem. Soc., 2009, 131, 5028; (c) F. Li, J. Zhang, X. Cao, L. Wang, D. Li, S. Song, B. Ye and C. Fan,
1846 | Analyst, 2014, 139, 1843–1846
15
16
17
18
19
20 21 22
Analyst, 2009, 134, 1355; (d) X. He, Z. Li, X. Jia, K. Wang and J. Yin, Talanta, 2013, 111, 105; (e) F. Jin, Y. Lian, J. Li, J. Zheng, Y. Hu, J. Liu, J. Huang and R. Yang, Anal. Chim. Acta, 2013, 799, 44; (f) S. Cheng, B. Zheng, M. Wang, M. H.-W. Lam and X. Ge, Talanta, 2013, 115, 506; (g) J. Chen and L. Zeng, Biosens. Bioelectron., 2013, 42, 93; (h) A. K. Sharma, A. D. Kent and J. M. Heemstra, Anal. Chem., 2012, 84, 6104; (i) X. Xu, J. Zhang, F. Yang and X. Yang, Chem. Commun., 2011, 47, 9435; (j) J. Chen, Z. Fang, P. Lie and L. Zeng, Anal. Chem., 2012, 84, 6321; (k) J. Lu, M. Yan, L. Ge, S. Ge, S. Wang, J. Yan and J. Yu, Biosens. Bioelectron., 2013, 47, 271; (l) J. Liu, Y. Liu, X. Yang, K. Wang, Q. Wang, H. Shi and L. Li, Anal. Chem., 2013, 85, 11121. (a) D. Chen, H. Feng and J. Li, Chem. Rev., 2012, 112, 6027; (b) C. Chung, Y.-K. Kim, D. Shin, S.-R. Ryoo, B. H. Hong and D.-H. Min, Acc. Chem. Res., 2013, 46, 2211. (a) C. H. Lu, H. H. Yang, C. L. Zhu, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2009, 48, 4785; (b) S. He, B. Song, D. Li, C. Zhu, W. Qi, Y. Wen, L. Wang, S. Song, H. Fang and C. Fan, Adv. Funct. Mater., 2010, 20, 453; (c) X. Liu, R. Aizen, R. Freeman, O. Yehezkeli and I. Willner, ACS Nano, 2012, 6, 3553. (a) H. Chang, L. Tang, Y. Wang, J. Jiang and J. Li, Anal. Chem., 2010, 82, 2341; (b) L. Wang, J. Zhu, L. Han, L. Jin, C. Zhu, E. Wang and S. Dong, ACS Nano, 2012, 6, 6659; (c) M. Zhang, B. Yin, W. Tan and B. Ye, Biosens. Bioelectron., 2011, 26, 3260. (a) C. H. Lu, J. A. Li, M. H. Lin, Y. W. Wang, H. H. Yang, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2010, 49, 8454; (b) Y. Wang, Z. Li, D. Hu, C.-T. Lin, J. Li and Y. Lin, J. Am. Chem. Soc., 2010, 132, 9274; (c) Y. Wang, Z. Li, T. J. Weber, D. Hu, C.-T. Lin, J. Li and Y. Lin, Anal. Chem., 2013, 85, 6775; (d) P. Zhang, Y. Wang, F. Leng, Z. H. Xiong and C. Z. Huang, Talanta, 2013, 112, 117. (a) Y. Wen, F. Xing, S. He, S. Song, L. Wang, Y. Long, D. Li and C. Fan, Chem. Commun., 2010, 46, 2596; (b) X. Zhao, R. Kong, X. Zhang, H. Meng, W. Liu, W. Tan, G. Shen and R. Yu, Anal. Chem., 2011, 83, 5062; (c) W. Y. Xie, W. T. Huang, N. B. Li and H. Q. Luo, Chem. Commun., 2012, 48, 82; (d) M. Li, X. Zhou, S. Guo and N. Wu, Biosens. Bioelectron., 2013, 43, 69; (e) Y. Yu, Y. Liu, S. J. Zhen and C. Z. Huang, Chem. Commun., 2013, 49, 1942. J. Liu, C. Wang, Y. Jiang, Y. Hu, J. Li, S. Yang, Y. Li, R. Yang, W. Tan and C. Z. Huang, Anal. Chem., 2013, 85, 1424. (a) D. Liao, H. Jiao, B. Wang, Q. Lin and C. Yu, Analyst, 2012, 137, 978; (b) W. C. Xu and Y. Lu, Anal. Chem., 2010, 82, 574. A. D. Kent, N. G. Spiropulos and J. M. Heemstra, Anal. Chem., 2013, 85, 9916.
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