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Cite this: Chem. Commun., 2014, 50, 180 Received 28th September 2013, Accepted 23rd October 2013 DOI: 10.1039/c3cc47418f www.rsc.org/chemcomm
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Label-free and ultrasensitive fluorescence detection of cocaine based on a strategy that utilizes DNA-templated silver nanoclusters and the nicking endonuclease-assisted signal amplification method† Kai Zhang,* Ke Wang, Xue Zhu, Jue Zhang, Lan Xu, Biao Huang and Minhao Xie*
A general and reliable strategy for the detection of cocaine was proposed utilizing DNA-templated silver nanoclusters as signal indicators and the nicking endonuclease-assisted signal amplification method. This strategy can detect cocaine specifically with a detection limit as low as 2 nM by using a small volume of 5 lL.
Aptamer, derived from the Latin word aptus meaning ‘‘to fit’’ and the Greek word meros meaning ‘‘part’’, has been a hot topic in recent years.1 Aptamers are nucleic acids with specific recognition properties toward their ligands. These DNA or RNA molecules, with unprecedented advantages such as synthetic convenience, chemical stability, easy modification, and so on, have become increasingly important molecular tools for diagnostics and therapeutics.2 In particular, aptamers have been popularly employed in the design of novel assay methods for small molecules,3 metal ions,4 and proteins,5 involving various signal-transduction approaches such as optical or electrochemical sensors. Albeit substantial progress was accomplished, a major disadvantage of homogeneous fluorescent aptamers is their relatively low association constants with substrates, which lead to low assay sensitivity. Thus, the development of amplification methods for homogeneous aptamer-based sensors (aptasensors) is essential. These techniques include the use of autonomous aptamer-based machines,6 the rolling circle amplification process,7 isothermal circular strand-displacement polymerization,8 and so on.9 However, these technologies always require the use of labelled fluorophores (donors) and quenchers (acceptors) for tagging, and quantum dots (QDs) or nanoparticles, which inevitably results in complicated or expensive operation. Therefore, the development of a label-free amplification strategy for homogeneous aptasensor that is simple and more sensitive is urgently required.
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China. E-mail: [email protected], [email protected]; Fax: +86-510-85520770; Tel: +86-510-85514482 † Electronic supplementary information (ESI) available: Experimental details and a figure showing the structures of DNA1 and the cocaine–DNA1–DNA2–DNA3 complex. See DOI: 10.1039/c3cc47418f
180 | Chem. Commun., 2014, 50, 180--182
Few-atom noble nanoclusters, such as silver nanoclusters (AgNCs), have been developed as a new class of fluorescent probes.10 Cytosine (C) rich nucleic acids usually act as stabilizing templates for AgNCs.11 The AgNCs consist of a few silver atoms and exhibit interesting photophysical properties.12 Indeed, these new fluorescent materials have been successfully applied in many research fields such as DNA sensing,13 protein assay,14 and cell imaging.15 Nicking endonucleases are a special family of restriction endonucleases that can recognize a specific sequence on a double-stranded DNA (dsDNA) and cut one strand of a dsDNA.16 This function has been used to develop many nicking endonuclease-based amplification strategies for the detection of different analytes.17 Some techniques based on nicking endonucleases are used for development of homogeneous fluorescence9 or quantum-dot based18 sensors. However, most of these nicking endonuclease-based sensors require the additional modification of aptamer probes or signaling DNA probes, which results in complicated or expensive operation. Enlightened by the above facts, we envision that a new strategy can be devised utilizing the advantages of both the nicking endonuclease-assisted signaling amplification (NEASA) strategy and the label-free architecture of AgNCs. This method does not need any chemical modification of DNA; the target molecule can be simply detected using the Microplate Readers, which makes it a simple and cost-effective method. With the use of cocaine as a proof-of-principle analyte, this sensing platform exhibited high sensitivity and specificity toward small molecules versus other nontargeted molecules. The working principle of the designed NEASA and AgNC-based aptasensor is illustrated in Scheme 1. The system mainly consists of DNA1, DNA2, and DNA3. The sequences of the oligonucleotides are listed in Fig. 1. DNA1 includes four domains: a sequence that is complementary to DNA2 and also contains the Nb.BbvCI recognition sequence (green domain), a linker part (black domain), a hybridization part (yellow domain) and the aptamer sequence for cocaine (magenta domain). The cocaine aptamer sequence was synthesized following the method reported by Cekan.19 DNA2 includes two AgNC-templatesegments (wine-colored domains), the recognition sequence and cleavage site (green domain) for Nb.BbvCI, and the hybridization
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Scheme 1 Schematic illustration of the NEASA and AgNC-based aptasensor for the detection of cocaine.
Fig. 1 DNA oligonucleotides sequence used in this strategy. The colors of the sequences are the same as given in Scheme 1.
part (yellow domain). DNA3 is designed to be partly complementary to both ends of DNA2. The hybridization of DNA2 and DNA3 results in DNA2 forming a closed structure. This closed structure holds the AgNC-template-segment in a rigid stage, which cannot combine with AgNCs, which results in very weak fluorescence. In the absence of cocaine, DNA3 hybridizes with DNA2, and forms a quasi-circular structure. As a result, the AgNC-template-segment is prevented from combining with the AgNCs and fold into a DNA–AgNC structure. When the target cocaine is introduced into the system, the hairpin structure is opened due to the specific binding of cocaine with its aptamer, thus facilitating the hybridization between DNA1 and the DNA2–DNA3 duplex structure to form a DNA1–DNA2–DNA3 complex. The formation of the DNA complex triggers the selective enzymatic cleavage of the DNA2–DNA3 duplex by Nb.BbvCI, resulting in the release of the AgNC-template-segments and blocker DNA. The released cocaine combined with DNA1 then hybridizes with another DNA2–DNA3 duplex structure to initiate the cleavage of the DNA2– DNA3 duplex structure, liberating the AgNC-template-segments and the release of blocker DNA. Eventually, each cocaine combined DNA1 can undergo many cycles, resulting in the digestion of many quasicircular DNA structures, generating many AgNC-template-segments. After amplification, the reporter oligonucleotide, the AgNC-templatesegment, acts as a scaffold for the synthesis of fluorescent silver nanoclusters in the presence of Ag+ through the reduction of NaBH4. The DNA–AgNCs scaffolded with the reporter oligonucleotide displayed fluorescence emission at 616 nm upon excitation at 530 nm. To verify the feasibility of quantitative detection, the fluorescence changes in the red-emission of AgNCs under different conditions were investigated. As shown in Fig. 2, the emission (at 616 nm) of
This journal is © The Royal Society of Chemistry 2014
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Fig. 2 The fluorescence emission spectra of AgNCs under different conditions: (a) DNA4 and DNA5; (b) 50 nM cocaine, DNA1, DNA2–DNA3 duplex, and Nb.BbvCI; (c) 50 nM cocaine, DNA1, and DNA2–DNA3 duplex; (d) DNA1, DNA2–DNA3 duplex, and Nb.BbvCI; (e) DNA1 and DNA2–DNA3 duplex; (f) DNA2–DNA3 duplex; (g) DNA1.
DNA1 (curve g), the DNA2–DNA3 duplex (curve f), and the mixture of DNA1 and DNA2–DNA3 duplex without (curve e) and with (curve d) Nb.BbvCI was relatively low. Upon addition of 50 nM cocaine to the above mixture without Nb.BbvCI, the emission intensity increase was only 41% (curve c). However, when both cocaine and Nb.BbvCI were present in the solution, we observed 996% increase in the emission intensity (curve b). This demonstrated that the fluorescence increase was attributed to the Nb.BbvCI activity. Thus, the proposed assay strategy could be used for amplified detection of cocaine. AgNCtemplate-segments (DNA4 and DNA5), two parts cut from DNA2 at the cleavage site of Nb.BbvCI, were employed for the control experiment (Fig. 1). The formation of DNA–AgNCs resulted in a high fluorescence signal intensity (curve a). The different concentrations of cocaine from a stock solution were added to the solution, and the resultant products of the NEASA reaction then acted as scaffolds for the synthesis of fluorescent AgNCs following reduction of Ag+ ions by NaBH4. Fig. 3A shows the emission spectra of the resultant DNA–AgNCs from the different concentrations of cocaine. As expected, a gradual increase in the fluorescence peak intensity at 616 nm was clearly observed with the increasing concentration of cocaine from 2 nM to 50 mM. From Fig. 3B, it can be seen that the fluorescence intensity increase (F0–F) is
Fig. 3 (A) The fluorescence emission spectra of the strategy for the assay of cocaine at different concentrations: from (a) to (o): 0, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 000, 20 000, and 50 000 nM cocaine, respectively. (B) The relationship between the fluorescence emission intensity increase and the logarithms of the concentrations of cocaine. The linear range is from 2 to 50 000 nM.
Chem. Commun., 2014, 50, 180--182 | 181
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Fig. 4 Selectivity tests of this assay, using cocaine and two analogues (ecgonine and benzoyl ecgonine) (0.5 mM each).
sensitive to the concentration of cocaine, the fitting range is from 2 nM to 50 mM with an equation Y = 270.92 X 68.83, where Y is the fluorescence intensity increase (F–F0) and X is the concentration of cocaine (regression coefficient R 2 = 0.998). The limit of detection of cocaine was 2 nM. To the best of our knowledge, it is one of the most sensitive methods for detection of cocaine. The detection sensitivity of the current assay can be significantly improved by 2 orders of magnitude over the previously reported AgNC-based assay for cocaine (100 nM),20 better by at least 3 orders of magnitude compared to the fluorescent sensor method (5 and 10 mM),6 and better by 4 orders of magnitude compared to the electrochemical method (10 mM).21 More significantly, our sensitive strategy meets the U.S. government’s guideline cutoff level established by the Substance Abuse & Mental Health Services Administration (SAMHSA) (300 ng mL 1 or 1 mM for the initial test, and 150 ng mL 1 or 0.5 mM for the confirming test).22 The specificity of this assay was also investigated by examining whether other common small molecules interfered with the assay for cocaine. Two cocaine analogues (0.5 mM each), ecgonine and benzoyl ecgonine, were chosen to be tested under the same experimental conditions as those used for cocaine (Fig. 4). They produced signals only slightly larger than the background, which was markedly lower than that for 0.5 mM of cocaine. These results suggest good selectivity of the assay for cocaine. The good selectivity probably resulted from the specific and high affinity binding of the aptamer to the target and the subsequent hybridization of DNA1 and DNA2. It is critically important to evaluate the real applicability of NEASA by challenging this strategy in various media. Serum is a complicated biological fluid containing a large number of proteins and other interfering materials. Given that the strategy is resistant to protein influence, we challenged the assay by analyzing samples of a complicated matrix to evaluate the applicability of the strategy to serum. Three concentrations of cocaine (10 nM, 100 nM, and 1000 nM) were spiked into 10-fold diluted human serum. The recovery values were determined and were acceptable (Table S1, ESI†). Therefore, our proposed NEASA assay could potentially be applied to realistic biological samples, such as diluted human serum. In summary, we have developed a novel highly sensitive method to quantify cocaine concentration as low as 2 nM by NEASA coupled with fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility in the application of detection of cocaine in real samples. This
182 | Chem. Commun., 2014, 50, 180--182
Communication
strategy has several excellent features. First, the assay does not involve any chemical modification of DNA, which makes it simple and low-cost. Second, the assay is conducted in aqueous solution, and does not require troublesome separation procedures. Third, it has high sensitivity and specificity. To the best of our knowledge, it is one of the most sensitive methods for detection and analysis of cocaine. Fourth, the assay volume of the small molecules is only 5 mL, which can reduce the sample volume usage. Finally, the concept can easily be extended to construct a series of probes by simply changing the cocaine aptamer domain sequence of DNA1. To sum up, the experimental results demonstrated that this simple and cost-effective method has the potential to be used as a major tool for simultaneous ultrasensitive quantitative analysis of cocaine or other biomarkers in serum and supply valuable information for biomedical research and clinical diagnosis. This work was supported by the Social Development Fund of Jiangsu Province (BE2013614), the Grants from National Natural Science Foundation (81300787), the Natural Science Foundation of Jiangsu Province (BK2011168, BK2012105), the Technology Infrastructure Plan of Jiangsu Province-Technology Public Service Platform (BM2012066), the Social Development Fund of Wuxi (CSE01N1239) and the Youth Foundation of Jiangsu Institute of Nuclear Medicine.
Notes and references 1 Y. Zhu, P. Chandra and Y.-B. Shim, Anal. Chem., 2012, 85, 1058–1064. 2 K. Zhang, M. Xie, B. Zhou, Y. Hua, Z. Yan, H. Liu, L. N. Guo, B. Wu and B. Huang, Biosens. Bioelectron., 2013, 41, 123–128. 3 Z. Zhu, C. Ravelet, S. Perrier, V. Guieu, E. Fiore and E. Peyrin, Anal. Chem., 2012, 84, 7203–7211. 4 X. Zhu, J. Zhao, Y. Wu, Z. Shen and G. Li, Anal. Chem., 2011, 83, 4085–4089. 5 K. Zhang, X. Zhu, J. Wang, L. Xu and G. Li, Anal. Chem., 2010, 82, 3207–3211. 6 B. Shlyahovsky, D. Li, Y. Weizmann, R. Nowarski, M. Kotler and I. Willner, J. Am. Chem. Soc., 2007, 129, 3814–3815. 7 Z.-S. Wu, H. Zhou, S. Zhang, G. Shen and R. Yu, Anal. Chem., 2010, 82, 2282–2289. 8 Q. Guo, X. Yang, K. Wang, W. Tan, W. Li, H. Tang and H. Li, Nucleic Acids Res., 2009, 37, e20. 9 R. Duan, X. Zuo, S. Wang, X. Quan, D. Chen, Z. Chen, L. Jiang, C. Fan and F. Xia, J. Am. Chem. Soc., 2013, 135, 4604–4607. 10 P. Shah, A. Rorvig-Lund, S. Ben Chaabane, P. W. Thulstrup, H. G. Kjaergaard, E. Fron, J. Hofkens, S. W. Yang and T. Vosch, ACS Nano, 2012, 6, 8803–8814. 11 X. Liu, F. Wang, R. Aizen, O. Yehezkeli and I. Willner, J. Am. Chem. Soc., 2013, 135, 11832–11839. 12 W. Guo, J. Yuan and E. Wang, Chem. Commun., 2011, 47, 10930–10932. 13 Q. Cui, Y. Shao, K. Ma, S. Xu, F. Wu and G. Liu, Analyst, 2012, 137, 2362–2366. 14 K. Zhang, K. Wang, M. Xie, X. Zhu, L. Xu, R. Yang, B. Huang and X. Zhu, Biosens. Bioelectron., 2014, 52, 124–128. 15 J. Yin, X. He, K. Wang, Z. Qing, X. Wu, H. Shi and X. Yang, Nanoscale, 2012, 4, 110–112. 16 Z.-z. Zhang and C.-y. Zhang, Anal. Chem., 2012, 84, 1623–1629. ˇek and M. Bartosˇ´k, 17 E. Palec ı Chem. Rev., 2012, 112, 3427–3481. 18 Y. Zhang and C.-y. Zhang, Anal. Chem., 2011, 84, 224–231. ¨ . Jonsson and S. T. Sigurdsson, Nucleic Acids Res., 19 P. Cekan, E. O 2009, 37, 3990–3995. 20 Z. Zhou, Y. Du and S. Dong, Biosens. Bioelectron., 2011, 28, 33–37. 21 B. R. Baker, R. Y. Lai, M. S. Wood, E. H. Doctor, A. J. Heeger and K. W. Plaxco, J. Am. Chem. Soc., 2006, 128, 3138–3139. 22 Y. Wen, H. Pei, Y. Wan, Y. Su, Q. Huang, S. Song and C. Fan, Anal. Chem., 2011, 83, 7418–7423.
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Published on 24 October 2013. Downloaded by Dalhousie University on 30/06/2014 15:02:26.
COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 180 Received 28th September 2013, Accepted 23rd October 2013 DOI: 10.1039/c3cc47418f www.rsc.org/chemcomm
View Article Online View Journal | View Issue
Label-free and ultrasensitive fluorescence detection of cocaine based on a strategy that utilizes DNA-templated silver nanoclusters and the nicking endonuclease-assisted signal amplification method† Kai Zhang,* Ke Wang, Xue Zhu, Jue Zhang, Lan Xu, Biao Huang and Minhao Xie*
A general and reliable strategy for the detection of cocaine was proposed utilizing DNA-templated silver nanoclusters as signal indicators and the nicking endonuclease-assisted signal amplification method. This strategy can detect cocaine specifically with a detection limit as low as 2 nM by using a small volume of 5 lL.
Aptamer, derived from the Latin word aptus meaning ‘‘to fit’’ and the Greek word meros meaning ‘‘part’’, has been a hot topic in recent years.1 Aptamers are nucleic acids with specific recognition properties toward their ligands. These DNA or RNA molecules, with unprecedented advantages such as synthetic convenience, chemical stability, easy modification, and so on, have become increasingly important molecular tools for diagnostics and therapeutics.2 In particular, aptamers have been popularly employed in the design of novel assay methods for small molecules,3 metal ions,4 and proteins,5 involving various signal-transduction approaches such as optical or electrochemical sensors. Albeit substantial progress was accomplished, a major disadvantage of homogeneous fluorescent aptamers is their relatively low association constants with substrates, which lead to low assay sensitivity. Thus, the development of amplification methods for homogeneous aptamer-based sensors (aptasensors) is essential. These techniques include the use of autonomous aptamer-based machines,6 the rolling circle amplification process,7 isothermal circular strand-displacement polymerization,8 and so on.9 However, these technologies always require the use of labelled fluorophores (donors) and quenchers (acceptors) for tagging, and quantum dots (QDs) or nanoparticles, which inevitably results in complicated or expensive operation. Therefore, the development of a label-free amplification strategy for homogeneous aptasensor that is simple and more sensitive is urgently required.
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China. E-mail: [email protected], [email protected]; Fax: +86-510-85520770; Tel: +86-510-85514482 † Electronic supplementary information (ESI) available: Experimental details and a figure showing the structures of DNA1 and the cocaine–DNA1–DNA2–DNA3 complex. See DOI: 10.1039/c3cc47418f
180 | Chem. Commun., 2014, 50, 180--182
Few-atom noble nanoclusters, such as silver nanoclusters (AgNCs), have been developed as a new class of fluorescent probes.10 Cytosine (C) rich nucleic acids usually act as stabilizing templates for AgNCs.11 The AgNCs consist of a few silver atoms and exhibit interesting photophysical properties.12 Indeed, these new fluorescent materials have been successfully applied in many research fields such as DNA sensing,13 protein assay,14 and cell imaging.15 Nicking endonucleases are a special family of restriction endonucleases that can recognize a specific sequence on a double-stranded DNA (dsDNA) and cut one strand of a dsDNA.16 This function has been used to develop many nicking endonuclease-based amplification strategies for the detection of different analytes.17 Some techniques based on nicking endonucleases are used for development of homogeneous fluorescence9 or quantum-dot based18 sensors. However, most of these nicking endonuclease-based sensors require the additional modification of aptamer probes or signaling DNA probes, which results in complicated or expensive operation. Enlightened by the above facts, we envision that a new strategy can be devised utilizing the advantages of both the nicking endonuclease-assisted signaling amplification (NEASA) strategy and the label-free architecture of AgNCs. This method does not need any chemical modification of DNA; the target molecule can be simply detected using the Microplate Readers, which makes it a simple and cost-effective method. With the use of cocaine as a proof-of-principle analyte, this sensing platform exhibited high sensitivity and specificity toward small molecules versus other nontargeted molecules. The working principle of the designed NEASA and AgNC-based aptasensor is illustrated in Scheme 1. The system mainly consists of DNA1, DNA2, and DNA3. The sequences of the oligonucleotides are listed in Fig. 1. DNA1 includes four domains: a sequence that is complementary to DNA2 and also contains the Nb.BbvCI recognition sequence (green domain), a linker part (black domain), a hybridization part (yellow domain) and the aptamer sequence for cocaine (magenta domain). The cocaine aptamer sequence was synthesized following the method reported by Cekan.19 DNA2 includes two AgNC-templatesegments (wine-colored domains), the recognition sequence and cleavage site (green domain) for Nb.BbvCI, and the hybridization
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 24 October 2013. Downloaded by Dalhousie University on 30/06/2014 15:02:26.
Communication
Scheme 1 Schematic illustration of the NEASA and AgNC-based aptasensor for the detection of cocaine.
Fig. 1 DNA oligonucleotides sequence used in this strategy. The colors of the sequences are the same as given in Scheme 1.
part (yellow domain). DNA3 is designed to be partly complementary to both ends of DNA2. The hybridization of DNA2 and DNA3 results in DNA2 forming a closed structure. This closed structure holds the AgNC-template-segment in a rigid stage, which cannot combine with AgNCs, which results in very weak fluorescence. In the absence of cocaine, DNA3 hybridizes with DNA2, and forms a quasi-circular structure. As a result, the AgNC-template-segment is prevented from combining with the AgNCs and fold into a DNA–AgNC structure. When the target cocaine is introduced into the system, the hairpin structure is opened due to the specific binding of cocaine with its aptamer, thus facilitating the hybridization between DNA1 and the DNA2–DNA3 duplex structure to form a DNA1–DNA2–DNA3 complex. The formation of the DNA complex triggers the selective enzymatic cleavage of the DNA2–DNA3 duplex by Nb.BbvCI, resulting in the release of the AgNC-template-segments and blocker DNA. The released cocaine combined with DNA1 then hybridizes with another DNA2–DNA3 duplex structure to initiate the cleavage of the DNA2– DNA3 duplex structure, liberating the AgNC-template-segments and the release of blocker DNA. Eventually, each cocaine combined DNA1 can undergo many cycles, resulting in the digestion of many quasicircular DNA structures, generating many AgNC-template-segments. After amplification, the reporter oligonucleotide, the AgNC-templatesegment, acts as a scaffold for the synthesis of fluorescent silver nanoclusters in the presence of Ag+ through the reduction of NaBH4. The DNA–AgNCs scaffolded with the reporter oligonucleotide displayed fluorescence emission at 616 nm upon excitation at 530 nm. To verify the feasibility of quantitative detection, the fluorescence changes in the red-emission of AgNCs under different conditions were investigated. As shown in Fig. 2, the emission (at 616 nm) of
This journal is © The Royal Society of Chemistry 2014
ChemComm
Fig. 2 The fluorescence emission spectra of AgNCs under different conditions: (a) DNA4 and DNA5; (b) 50 nM cocaine, DNA1, DNA2–DNA3 duplex, and Nb.BbvCI; (c) 50 nM cocaine, DNA1, and DNA2–DNA3 duplex; (d) DNA1, DNA2–DNA3 duplex, and Nb.BbvCI; (e) DNA1 and DNA2–DNA3 duplex; (f) DNA2–DNA3 duplex; (g) DNA1.
DNA1 (curve g), the DNA2–DNA3 duplex (curve f), and the mixture of DNA1 and DNA2–DNA3 duplex without (curve e) and with (curve d) Nb.BbvCI was relatively low. Upon addition of 50 nM cocaine to the above mixture without Nb.BbvCI, the emission intensity increase was only 41% (curve c). However, when both cocaine and Nb.BbvCI were present in the solution, we observed 996% increase in the emission intensity (curve b). This demonstrated that the fluorescence increase was attributed to the Nb.BbvCI activity. Thus, the proposed assay strategy could be used for amplified detection of cocaine. AgNCtemplate-segments (DNA4 and DNA5), two parts cut from DNA2 at the cleavage site of Nb.BbvCI, were employed for the control experiment (Fig. 1). The formation of DNA–AgNCs resulted in a high fluorescence signal intensity (curve a). The different concentrations of cocaine from a stock solution were added to the solution, and the resultant products of the NEASA reaction then acted as scaffolds for the synthesis of fluorescent AgNCs following reduction of Ag+ ions by NaBH4. Fig. 3A shows the emission spectra of the resultant DNA–AgNCs from the different concentrations of cocaine. As expected, a gradual increase in the fluorescence peak intensity at 616 nm was clearly observed with the increasing concentration of cocaine from 2 nM to 50 mM. From Fig. 3B, it can be seen that the fluorescence intensity increase (F0–F) is
Fig. 3 (A) The fluorescence emission spectra of the strategy for the assay of cocaine at different concentrations: from (a) to (o): 0, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 000, 20 000, and 50 000 nM cocaine, respectively. (B) The relationship between the fluorescence emission intensity increase and the logarithms of the concentrations of cocaine. The linear range is from 2 to 50 000 nM.
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Fig. 4 Selectivity tests of this assay, using cocaine and two analogues (ecgonine and benzoyl ecgonine) (0.5 mM each).
sensitive to the concentration of cocaine, the fitting range is from 2 nM to 50 mM with an equation Y = 270.92 X 68.83, where Y is the fluorescence intensity increase (F–F0) and X is the concentration of cocaine (regression coefficient R 2 = 0.998). The limit of detection of cocaine was 2 nM. To the best of our knowledge, it is one of the most sensitive methods for detection of cocaine. The detection sensitivity of the current assay can be significantly improved by 2 orders of magnitude over the previously reported AgNC-based assay for cocaine (100 nM),20 better by at least 3 orders of magnitude compared to the fluorescent sensor method (5 and 10 mM),6 and better by 4 orders of magnitude compared to the electrochemical method (10 mM).21 More significantly, our sensitive strategy meets the U.S. government’s guideline cutoff level established by the Substance Abuse & Mental Health Services Administration (SAMHSA) (300 ng mL 1 or 1 mM for the initial test, and 150 ng mL 1 or 0.5 mM for the confirming test).22 The specificity of this assay was also investigated by examining whether other common small molecules interfered with the assay for cocaine. Two cocaine analogues (0.5 mM each), ecgonine and benzoyl ecgonine, were chosen to be tested under the same experimental conditions as those used for cocaine (Fig. 4). They produced signals only slightly larger than the background, which was markedly lower than that for 0.5 mM of cocaine. These results suggest good selectivity of the assay for cocaine. The good selectivity probably resulted from the specific and high affinity binding of the aptamer to the target and the subsequent hybridization of DNA1 and DNA2. It is critically important to evaluate the real applicability of NEASA by challenging this strategy in various media. Serum is a complicated biological fluid containing a large number of proteins and other interfering materials. Given that the strategy is resistant to protein influence, we challenged the assay by analyzing samples of a complicated matrix to evaluate the applicability of the strategy to serum. Three concentrations of cocaine (10 nM, 100 nM, and 1000 nM) were spiked into 10-fold diluted human serum. The recovery values were determined and were acceptable (Table S1, ESI†). Therefore, our proposed NEASA assay could potentially be applied to realistic biological samples, such as diluted human serum. In summary, we have developed a novel highly sensitive method to quantify cocaine concentration as low as 2 nM by NEASA coupled with fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility in the application of detection of cocaine in real samples. This
182 | Chem. Commun., 2014, 50, 180--182
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strategy has several excellent features. First, the assay does not involve any chemical modification of DNA, which makes it simple and low-cost. Second, the assay is conducted in aqueous solution, and does not require troublesome separation procedures. Third, it has high sensitivity and specificity. To the best of our knowledge, it is one of the most sensitive methods for detection and analysis of cocaine. Fourth, the assay volume of the small molecules is only 5 mL, which can reduce the sample volume usage. Finally, the concept can easily be extended to construct a series of probes by simply changing the cocaine aptamer domain sequence of DNA1. To sum up, the experimental results demonstrated that this simple and cost-effective method has the potential to be used as a major tool for simultaneous ultrasensitive quantitative analysis of cocaine or other biomarkers in serum and supply valuable information for biomedical research and clinical diagnosis. This work was supported by the Social Development Fund of Jiangsu Province (BE2013614), the Grants from National Natural Science Foundation (81300787), the Natural Science Foundation of Jiangsu Province (BK2011168, BK2012105), the Technology Infrastructure Plan of Jiangsu Province-Technology Public Service Platform (BM2012066), the Social Development Fund of Wuxi (CSE01N1239) and the Youth Foundation of Jiangsu Institute of Nuclear Medicine.
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