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Silver Nanoclusters Beacon as Stimuli-Responsive Versatile Platform for Multiplex DNAs Detection and Aptamer-substrate Complexes Sensing Guoliang Liu, Jingjing Li, Da-Qian Feng, Jun-Jie Zhu, and Wei Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04362 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016
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Analytical Chemistry
Silver Nanoclusters Beacon as Stimuli-Responsive Versatile Platform for Multiplex DNAs Detection and Aptamer-substrate Complexes Sensing ⊥
⊥
Guoliang Liu,†,‡, Jingjing Li,§, Da-Qian Feng,‡ Jun-Jie Zhu,*,† Wei Wang*,‡ †
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. ‡School of Chemistry and Chemical Engineering, Yancheng Institute of Technology, ⊥ Yancheng 224051, China. §School of Medical Imaging, Xuzhou Medical University, Xuzhou, China. These authors contributed equally to this work. Fax/Tel: +86-25-8357204. *E-mail:
[email protected];
[email protected]. ABSTRACT: Activatable silver nanoclusters beacon (ASNCB) were synthesized through a facile one-pot approach and applied for multiplex DNAs, small molecule and protein sensing. Multifunctional single-stranded DNA sequences are rationally designed and used for ASNCB in-situ synthesis. Via target-responsive structure transformation of ASNCB, target recognition induced ASNCB conformational transition and light up the fluorescent signal of silver nanoclusters. By further implementing two different color ASNCB (520 nm and 600 nm), the parallel multiplexed analysis of two target genes (Influenza A virus genes H1N1 and H5N1) is achieved. Additionally, with the introduction of aptamer for the design of the molecular beacon, the detections of small molecule, ATP and biomacromolecule, thrombin have also been realized. It is for the first time to allow the generation of activatable fluorescent Ag NCs-based probe and the target recognition integrated into a single process, which provides a versatile platform for different analytes in a facile way. The successful application of our proposed ASNCB in real sample analysis and ATP imaging in living cells further displayed their potential promising for fluorescence sensing.
INTRODUCTION The development of sensitive and specific molecular probes is one of the central challenges in cancer imaging. Optical imaging probes have been developed well due to their excellent sensitivity. Particularly, activatable fluorescent molecular probes have been paid more attention due to the specific response to the target and better signal-to-noise.1-4 These activatable probes commonly consisted of a fluorescent dye and a quencher attached to the opposite ends of a peptide or polymer linker. Once the cleavage of the linker by cancerrelated enzyme, the dye would be separated from the quencher, inducing an activated signal. However, these methods cannot monitor targets in real-time due to the separation of the dye from the probe. Moreover, the dye as signal reporter suffered from toxicity, low biocompatibility, and photobleaching. Different from the dye, fluorescent nanomaterials such as quantum dot, noble nanoclusters have sub-nanometer, good photostability and tunable fluorescence. Therefore, it is promising to develop activatable fluorescent nanoprobe for disease-related target sensing. More recently, noble metal nanoclusters are attracting increasing attention from the research community for both basic and applied studies because of its possessing unique molecular-like properties, including strong fluorescence, welldefined molecular structures, quantized charging and so on.5,6 Noble metal nanoclusters comprise several to a few hundred metal atoms with a typical size of below 2 nm comparable to the Fermi wavelength of electrons, which induced discrete energy levels instead of continuous density of states, thereby
leading to distinctive molecular-like properties.7,8 Especially, nucleic acid-stabilized silver nanoclusters (DNA-Ag NCs) as a new kind of fluorophores have attracted the widespread interest of interdisciplinary research.9-10 The preparation of Ag NCs in aqueous solutions is difficult due to the tendency of Ag NCs to aggregate. The DNA molecule provides an ideal scaffold for the synthesis of Ag NCs because the structure of DNA plays a crucial role in the successful synthesis of Ag NCs as well as their fluorescence porperties.11 The DNA-Ag NCs exhibit unique properties such as good photostability, water solubility, biocompatibility, low toxicity, and tunable emission. Furthermore, the photophysical properties could be rationally controlled by changing the length or base sequence of the template, providing an easy way for the design of fluorescent probe.12 These features turned the Ag NCs to attractive materials for optical sensing and biological imaging applications.13-20 Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes that recognize the specific nucleic acids with allosteric effect occurring to control the distance between the dye and the quencher.21,22 In the recognize process, fluorescent dye still attaches to the probe, which makes MBs the potentiality to construct new activatable probe and for realtime monitoring. However, conventional MBs' applications are limited because of the requirement of the labeling with an appropriate fluorescent dye/quencher.16,23 Double labeling processes bring high cost of reagents and multiple steps of purification. Besides, developing new approach for the simultaneous detection of multiple targets is a great challenge in molecular diagnostics and imaging.24-27
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Scheme 1. Schematic representation of the novel strategy for one-pot synthesis of molecular beacon-based activatable Ag NCs for DNA and Aptamer sensing. Herein, original activatable silver nanoclusters beacon (ASNCB) were synthesized with multifunctional DNAs as the templates for the sensing of DNA, small molecule and protein, as illustrated in Scheme 1. To achieve this aim, multifunctional ssDNA which contains three different domains were rationally designed: the DNA scaffold for fluorescent Ag NC synthesis (Domain I), the recognition sequence to the target (Domain II), and a quencher attached sequence partially complemented to Domain II for the formation of molecular beacon structure (Domain III). With this multifunctional DNA as template, the prepared fluorescent Ag NCs and the quencher would get in close proximity because of the formation of molecular beacon structure. Thus, the fluorescence of Ag NCs was quenched obviously by the quencher based on fluorescence resonance energy transfer (FRET) mechanism. However, upon the specific recognization of the target to Domain II, the hairpin structure of molecular beacon would be destroyed and the Ag NCs were separated far from the quencher, generating an activated fluorescence signal. With the design of Domain II sequence, different targets could be detected, such as DNA, small molecule and protein. In this work, we realized the analysis of DNA, adenosine triphosphate (ATP) and thrombin with the introduction of aptamer. More importantly, the tunable fluorescence properties of Ag NCs enabled the multiplexed detection of Influenza A virus DNAs H1N1 and H5N1 simultaneously. As far as we know, this is first work integrating the construction of activatable fluorescence Ag NCs-based probe and the target recognition into a single process, which provides great opportunity for developing versatile platform in facile and rapid way.
EXPERIMENTAL SECTION Materials. Silver nitrate (AgNO3, 99.99%) and sodium borohydride (NaBH4, 98%) were purchased from Alfa Aesar and used without further purification. The DNA strands were purchased from Sangon (Shanghai, China). All other reagents were all of analytical grade and used as received without further purification. Millipore Milli-Q ultrapure water (18.2 MΩ. cm) was used for all experiments. The citrate buffer solution (20 mM, pH 7.0) was used to control the acidity of the reaction. All oligonucleotides were HPLC-purified and freeze-dried by the supplier. The oligonucleotides were used as provided and dissolved in an ultrapure water to give stock solutions of 100 µM. The sequences of the oligonucleotides that were used in this study are as follows: (1) 5'-CCCTTTAACCCC-3'
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(2) 5'-CCTCCTTCCTCC-3' (3) 5'-CCCTAACTCCCC-3' (4) 5'-CGACTACACTCTCGATGAAGAA-3' (5) 5'-CATACTGAGAACTCAAGAGTCT-3' (6) 5'-CCCTTTAACCCCTTCTTCATCGAGAGTGTAGTCG GAAGAA-3' (7) 5'-CCTCCTTCCTCCAGACTCTTGAGTTCTCAGTATG GAGTCT-3' (8) 5'-CCCTAACTCCCCAGACTCTTGAGTTCTCAGTATG GAGTCT-3' (9) 5'-CCCTTTAACCCCTTCTTCATCGAGAGTGTAGTCG GAAGAA-BHQ1-3' (10) 5'-CCTCCTTCCTCCAGACTCTTGAGTTCTCAGTATG GAGTCT-BHQ2-3' (11) 5'-CCCTAACTCCCCAGACTCTTGAGTTCTCAGTAT GGAGTCT-BHQ3-3' (12) 5'-CCTCCTTCCTCCTACCTGGGGGAGTATTGCGGA GGAAGGTTCCCCCAGG-3' (13) 5'-CCCTTTAACCCCTAGGTTGGTGTGGTTGGTGT GGTTGGACACCAACC-3' (14) 5'-CCTCCTTCCTCCTACCTGGGGGAGTATTGCGG AGGAAGGTTCCCCCAGG-BHQ2-3' (15) 5'-CCCTTTAACCCCTAGGTTGGTGTGGTTGGTGT GGTTGGACACCAACC-BHQ1-3' (16) 5'-CCTCCTTCCTCCTACCTGGGGGAGTATTGCGG AGGAAGGTCCCAGG-3' (17) 5'-CCTCCTTCCTCCTACCTGGGGGAGTATTGCGG AGGAAGGTCCCAGG-BHQ2-3' (18) 5'-CCCTTTAACCCCTAGGTTGGTGTGGTTGGTGT GGTTGGCCAACC-3' (19) 5'-CCCTTTAACCCCTAGGTTGGTGTGGTTGGTGT
GGTTGGCCAACC-BHQ1-3' Apparatus. Fluorescence spectra were carried out using a Hitch F-7000 fluorescence spectrophotometer. The greenemitting Ag NCs were excited at λ = 442 nm. The red-emitting Ag NCs were excited at λ = 530 nm. The near-infraredemitting Ag NCs were excited at λ = 710 nm. Synthesis of fluorescent silver nanoclusters. The 15 µM solution of hairpin oligonucleotide probes were diluted with 20 mM citrate buffer (pH 7.0) and were heated at 95 °C for 10 min. Then, the solution annealed quickly in the ice bath for at least 1 hour. After that, a 90 µM Ag NO3 was added into the MBA-Ag NCs probe solution, followed by the vigorous shaking for 2 min. After the continue ice bath for 15 min, the 90 µM freshly prepared Na BH4 was added to the solution, followed by vigorous shaking of the mixture for 5 min. The solution was kept in the dark at room temperature and was allowed to react for 12 h before the measurement. Analysis of Target DNAs using ASNCB. In the typical DNA assay, the synthesized ASNCB were hybridized with different concentrations of target DNAs in citrate buffer (20 mM, pH 7.0). The fluorescence spectra of ASNCB were measured. Multiplexed Fluorescent DNAs detection. For the multiplexed analysis of Influenza A virus DNAs, greenemitting 9 ASNCB and red-emitting 10 ASNCB were mixed together. The respective target DNAs were then added, and the time-dependent fluorescence changes of SNCB were recorded. For the multiplexed detection of DNA 4 (500 nM) and of DNA 5 (500 nM), green-emitting 9 ASNCB probe and redemitting 10 ASNCB probe were selected. Detection of ATP or Thrombin using Fluorescent ASNCB. The Ag NCs-linked ATP aptamer or the Ag NCslinked thrombin aptamer was mixed with different concentrations of ATP or thrombin, respectively. The
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activatable fluorescence spectra resulting upon the formation of the respective aptamer-substrate complexes were monitored.
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Figure 1. (a) Emission spectra of synthesized ssDNA-Ag NCs: 1 (black line), 2 (red line), and 3 (blue line), respectively. (b) Emission spectra of pre-prepared silver nanoclusters beacon (SNCB): 6 (black line), 7 (red line), and 8 (blue line), respectively. In order to obtain an activated fluorescent response to the targets, the fluorescence property of Ag NCs stabilized by the multifunctional DNA should be concerned first. Three different Ag NCs stabilized with single-strand DNA 1, 2, and 3 with the maximum wavelength at 520 nm, 608 nm, and 740 nm, were applied in this work (Figure 1a). Influenza A virus H1N1 gene and Influenza A virus H5N1 gene were chosen as the target models, named as DNA 4 and 5. H1N1 and H5N1 genes are the subtypes of Influenza A virus which can cause illness in humans and many other animal species. As shown in Scheme 1, DNA 1, 2, 3 were employed as Domain I (colored bases) and elongated with the target recognition sequence (Domain II, underlined bases), and a sequence partially complement to Domain II (Domain III) (Please see detailed explanations in Experimental Section). The obtained multifunctional DNA sequences were named as 6, 7 and 8 to stabilize fluorescent Ag NCs, where multifunctional DNA 6 was designed to target DNA 4, and DNA 7, 8 were designed to target DNA 5. As shown in Figure 1b, multifunctional DNA 6, 7, and 8 stabilized Ag NCs displayed the maximum wavelength at 520, 600, 740 nm, almost the same with those of ssDNA 1, 2 and 3-templated Ag NCs. It indicated that such elongation did not affect the fluorescence emission of Ag NCs, providing a basis for the fabrication of activated fluorescent probe. The HR-TEM images of the prepared Ag NCs were monodispersed with a size of 2 nm (Figure S1). Besides, the stability experiment of the as-synthesized fluorescent probes was also carried out. The assay results revealed that our synthesized DNA-Ag NCs kept nearly same fluorescence intensity with six months as new synthesis sample (Figure S2). Then, to further construct the activatable fluorescent probe, nucleic acid 6, 7, 8 were modified with a quencher, BHQ molecule, at 3'-end, and named as 9, 10, 11 accordingly. After the thermal denaturation and annealing process (Scheme S1), molecular beacon structure formed DNA 9, 10, 11 were used as template to prepare fluorescent Ag NCs (9 ASNCB, 10 ASNCB, 11 ASNCB) (Scheme S2). Taking 9 ASNCB for example, the presence of BHQ molecules and their close proximity to Ag NCs quenched the fluorescence of Ag NCs obviously. But with the introduction of their target gene, DNA 4, its hybridization with Domain II in DNA 9 opened the molecular beacon structure and separated Ag NCs from the quencher BHQ1 molecule, producing an activated fluorescence signal of 9 ASNCB (Figure S3). As the
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Figure 2. (a) Assay of the target DNAs using activatable silver nanoclusters beacon (ASNCB) probe as sensing matrices. (b) Time-dependent fluorescence changes of the green-emitting 9 ASNCB probe at λ = 507 nm after the introduction of target DNA 4 (500 nM). (c) Fluorescence spectra of 9 ASNCB probe in absence and presence of different concentrations of target gene 4. Conditions: 1, 9 ASNCB probe; 2-10, 1 + CDNA 4 (nM): 10, 25, 50, 100, 250, 500, 1000, 1500, and 2000. Fluorescence spectra were recorded after a fixed time interval of 30 min. The inserted figure is the calibration curve corresponding to the fluorescence of the related 9 ASNCB (λ = 507 nm) in the presence of different concentrations of target DNA 4. (d) Time-dependent fluorescence changes of the red-emitting 10 ASNCB probe at λ = 598 nm after the introduction of target DNA 5 (500 nM). (e) Fluorescence spectra of 10 ASNCB probe in absence and presence of different concentrations of target DNA 5. Conditions: 1, 10 ASNCB probe; 2-9, 1 + CDNA 5 (nM): 25, 50, 100, 250, 500, 1000, 1500, and 2000. The inserted figure is the calibration curve corresponding to the fluorescence of the 10 ASNCB probe (λ = 598 nm) in the presence of different concentrations of target DNA 5. F0 and F correspond to the fluorescence intensity of the system in the absence and presence of target DNA, respectively. Error bars were derived from N = 3 experiments. separation of Ag NCs from the quencher molecule was controlled by the concentration of the target DNA 4, the approach was anticipated to provide a quantitative means to analyze the target gene (Figure 2a). To look for an optimal time-interval to record the activated fluorescent signal, the time-dependent fluorescence changes upon subjecting target DNA 4 were studied. As shown in Figure 2b, the fluorescence of green-emitting 9 ASNCB was increased with the time passing, suggesting that the Ag NCs was separated from the quencher BHQ1 molecule. The fluorescence reached saturated after approximately 30 min. Thus, the fluorescent signals were collected with the different
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Figure 3. Fluorescence intensity enhancement (F-F0/F0) of 9 ASNCB probe at 507 nm (a) and 10 ASNCB probe at 598 nm (b) in the presence of 500 nM complementary target DNA (DNA 4, H1N1DNA; DNA 5, H5N1DNA), single-base mismatch DNA (SMDNA) and random DNA(RDNA). F0 and F correspond to the fluorescence intensity of the detected system in the absence and presence of target DNAs, respectively. Error bars were obtained from three parallel experiments. In order to investigate the specificity of 9 ASNCB or 10 ASNCB probe to target DNAs 4 or 5, their fluorescence changes in the presence of the single-base mismatch DNA (SM1DNA and SM2DNA) and random DNA (RDNA1 and RDNA2) with the concentration of 500 nM were studied under the same experimental conditions. As shown in Figure 3a, the fluorescence enhancement (F-F0)/F0 were only 0.3 and 0.09 after the introduction of SM1DNA and RDNA1, which were much lower than that of target DNA 4, 1.0. Similarly, the fluorescence enhancement of 10 ASNCB probe in the presence of SM2DNA and RDNA2 were 0.39, and 0.1 (Figure 3b), indicating the excellent selectivity for the target genes even in the single nucleotide mutation. Multiplexed optical DNA sensing is a challenging topic in the bioanalysis field due to the need of multiple labeling of different signal reporters. To explore the possibility of our fabricated ASNCB probe for the multiplexed detection of target genes, the green-emitting 9 ASNCB probe and the redemitting 10 ASNCB probe were used to sense target DNA 4 and 5 simultaneously (Figure 4a). Only very weak fluorescence emissions were detected in the absence of two
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concentrations of target gene 4 at a fixed time-interval of 30 min. As shown in Figure 2c, with the concentration of target gene 4 increased, the fluorescence signals of the activated green-emitting 9 ASNCB were intensified, presenting a dynamic range from 10 nM to 1000 nM and a detection limit of 2 nM. The corresponding UV-vis spectra were also monitored as listed in Figure S4a, showing a decreased absorbance at 536 nm. Similarly, good performance of the red-emitting 10 ASNCB for the detection of the Influenza A virus H5N1 DNA 5 were also obtained (Figure 2d and 2e). The nucleic acid sequence (domain I) protecting red-emitting Ag NCs was elongated with the complementary sequence to DNA 5 (domain II) and the short DNA sequence complementary to a part of the domain II modified with the quencher BHQ2 molecule. The quenched fluorescence of Ag NCs was triggered on upon the introduction of DNA 5. As shown in Figure 2e, a dynamic range from 25 nM to 1000 nM and a detection limit of 10 nM for target DNA 5 was presented. The UV-vis spectra of 10 ASNCB in the absence and presence of increasing concentrations of target DNA 5 were also carried out (Figure S4b). The results exhibited an enhanced absorbance at 585 nm which kept accordance with the fluorescence emission spectra.
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Figure 4. (a) Multiplexed detection of target DNAs 4 and 5 using green-emitting 9 and red-emitting 10 ASNCB probes. Fluorescence spectra of green-emitting 9 ASNCB probe and red-emitting 10 ASNCB probe for multicolor detection in the absence of the target genes (DNA 4 and DNA 5) (b). In the presence of 500 nM DNA 4 and absence of DNA 5 (c). In the absence of DNA 4 and presence of 500 nM DNA 5 (d). In the presence of 500 nM DNA 4 and 500 nM DNA 5 (e). Curve a (the black line) and curve b (the red line) correspond to fluorescence emission spectrum of green-emitting 9 ASNCB probe and red-emitting 10 ASNCB probe, respectively. Fluorescence spectra were recorded after a fixed time interval of 30 min. genes (Figure 4b). But after the introduction of DNA 4 or DNA 5, the conformation change activated the fluorescence signal of 9 or 10 ASNCB probe (Figure 4c or 4d). Additionally, such responses were not affected in the coexistence of DNA 4 and DNA 5 (Figure 4e), displaying their promising for the simultaneous detection of multiple targets. a hv
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Scheme 2. (a) Assay of ATP using 12 SNCB or 14 ASNCB probes. (b) Assay of thrombin using 13 SNCB or 15 ASNCB probes.
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Furthermore, ASNCB probes might be extended for the detection of the aptamer-substrate complexes with the introduction of aptamer for the multifunctional DNA design. To testify this hypothesis, ATP and thrombin aptamers were included as Domain II for the design of multifunctional DNAs (DNA 12 for ATP and DNA 13 for thrombin) (Scheme 2). Considering steric hindrance, a random TA base spacer was chosen for the separation of the Ag NCs template from aptamer sequence. Accordingly, BHQ2 and BHQ1 attached DNA 12 and 13 were named as DNA 14 and 15. DNA 12 stabilized Ag NCs (12 SNCB) presented red color emissions (Figure S5a), while a fluorescent quenched signal was observed for 14 ASNCB due to the formation of molecular beacon (Figure S5b). In the presence of ATP, its binding with aptamer opened the loop of molecular beacon, which kept the silver nanoclusters away from the quencher BHQ2 molecule. This separation activated the fluorescence signal of 14 ASNCB probe and the fluorescence was gradually intensified with the increase of ATP concentration (Figure 5a). The
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Figure 5. (a) Fluorescence spectra of red-emitting Ag NCs upon detecting different concentrations of ATP by 14 ASNCB probe. Conditions: The concentrations of ATP (mM): 1-6: 0, 0.05, 0.25, 0.5, 1, 2. Fluorescence spectra were recorded after a fixed time interval of 30 min. Inset: calibration curve corresponding to the fluorescence signal at λ = 568 nm. Error bars were derived from N = 3 experiments. (b) Selectivity of ATP aptasensor by using 1 mM of UTP, GMP, GTP, and ATP. (c) Fluorescence spectra of green-emitting 15 ASNCB probe upon detecting different concentrations of thrombin. Conditions: The concentrations of thrombin: (1) 0, (2) 5 nM, (3) 25 nM, (4) 50 nM, (5) 75 nM, (6) 100 nM. Fluorescence spectra were recorded after a fixed time interval of 30 min. Inset: calibration curve corresponding to the luminescence signal at λ = 517 nm. Error bars were derived from N = 3 experiments. (d) Time-dependent fluorescence changes upon detecting 75 nM thrombin by 15 ASNCB probe (1, black curve) and 19 ASNCB probe (2, red curve). (e) Confocal fluorescence images of MCF-7 cells treated with (I) The control probe, (II) 14 ASNCB (red), and (III) MitoTracker®Green (green). (IV) Overlap of the corresponding fluorescence images.
method enabled ATP analysis with a detection limit of 2 µM. The linear ranges change from 10 µM to 500 µM. To investigate its specificity, the fluorescence responses of 14 ASNCB probe to other similar targets such as uridine triphosphate (UTP), guanosine monophosphate (GMP), and guanosine triphosphate (GTP) were studied. The results revealed that 14 ASNCB probe did not respond to them (Figure 5b). Moreover, DNA 16 is a multifunctional DNA sequence similar with DNA 12 except six bases stem instead of nine bases stem. BHQ2 attached DNA 16 were named as DNA 17. The experimental results suggested that 14 ASNCB had better analytical performance than 17 ASNCB (Figure S6). Moreover, the MBA-Ag NCs fluorescent probe could be further implemented for the detection of macromolecules, such as thrombin protein by the replacement of ATP aptamer with thrombin aptamer (Scheme 2b). As mentioned above, the nucleic acid 15 was composed of the DNA template for the green-emitting Ag NCs, a random TA base spacer, and a BHQ1 modified short DNA sequence complementary to part of the aptamer. Similar with ATP detection, DNA 13 without the quencher BHQ1 modification could stabilize fluorescence Ag NCs with a 568 nm emissions (Figure S7a), while in the presence of BHQ1, the fluorescence of 15 ASNCB was quenched obviously (Figure S7b). Such quenched fluorescence was activated with the introduction of thrombin, and intensified with the increased concentration of thrombin, which enabled the determination of thrombin with a detection limit of 0.5 nM (Figure 5c). The linear ranges were 2 nM ~ 300 nM. In order to obtain the probe with the optimal signalto-background ratio, different stem lengths (nine bases and six bases) were designed. DNA 18 was similar with DNA 13 except six bases stem instead of nine bases stem. BHQ1 attached DNA 18 called DNA 19. Figure 5d showed the timedependent fluorescence changes to thrombin (75 nM) by the 15 ASNCB probe (black curve) and 19 ASNCB probe (red curve). The results implied that the stem length of molecular beacon based probe might be an important parameter for the optimization of the sensor performance. To demonstrate the capacity of developed SNCB in ATPtargeted imaging, 14 ASNCB was as a model for treating with MCF-7 cancer cells. After incubation with 14 ASNCB or MitoTracker®Green for 4 h, strong red fluorescence signals were detected from MCF-7 cells by confocal microscopy imaging (Figure 5eII). A negative control experiment performed with random chain instead of ATP aptamer in 14 ASNCB, the fluorescence recovery was not observed (Figure 5eI). By comparing with mitochondria staining Mitro Tracker®Green, it was observed that 14 ASNCB was mainly accumulated in cytoplasm of MCF-7 cells (Figure 5eIII and eIV). These phenomena suggest that our proposed activatable silver nanoclusters beacon sensing platform has the potentiality of ATP imaging in living cells. The developed molecule beacon-based activatable Ag NCs provided an effective fluorescence probe for the detection of DNA or aptamer-substrate complexes. Their performance comparison with other activatable optical methods reported in the literature was listed in Table S1, S2 and S3.28-39 For DNA detection, our proposed ASNCB revealed better or comparable sensitivity as other detection methods (Table S1). Similarly, for the determination of ATP or thrombin by other aptamerbased sensors, the developed SNCB also exhibited comparable sensitivity (Table S2 and Table S3). To assess the application of the present ASNCB sensor in complex biological systems,
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the detections of ATP in human urine and thrombin in human serum samples were carried out. The results provided by the proposed ASNCB sensor were compared with those obtained by the standard HPLC method. As shown in Table S4 and S5, good consistency was observed between these two methods, indicating the excellent behavior of our proposed ASNCB sensor in real sample analysis.
CONCLUSION In summary, the molecular beacon-based activatable fluorescent Ag NCs sensing system was developed for the fluorescent detection of DNAs and aptamer-substrate complexes. Besides, multiplexed detection of genes were also realized with the employment of different color fluorescent ASNCB. Compared with reported activatable probes, our proposed ASNCB probe owns some unique features: (1) The original ASNCB was successfully synthesized by rationally design, which firstly integrated the generation of light-up fluorescent Ag NCs and the target recognition into a single process. (2) The design of multifunctional ssDNA sequence for constructing activatable fluorescent ASNCB probe avoids troublesome fluorophore dyes labeling procedure and function modification of recognition molecules. (3) The developed ASNCB exhibit activatable fluorescent signal in the presence of targets without the cleavage of the fluorescent reporter from the quencher, which can be applied in real-time monitoring in vitro and in vivo. (4) The present sensing probe can be used for the detection of multiple targets with only change the recognition sequence. (5) The developed ASNCB probe is demonstrated for simultaneously multiplex detecting gene DNAs. Moreover, the present study paves the way to develop novel activatable probe for the multiplexed determination of DNAs or aptamer-substrate complexes. Furthermore, the successful application of our present ASNCB in real sample analysis and ATP imaging in living cells further revealed their potential promising for fluorescence sensing in complicated bio-samples.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The HRTEM images of 1 ssDNA-Ag NCs or 6 SNCB, UV-vis spectra of 9 and 10 ASNCB probe, fluorescence spectra of 9 ASNCB, 12 SNCB, and 13 SNCB, time-dependent fluorescence changes upon detecting 1 mM ATP by 14 ASNCB probe (1, black curve) or 17 ASNCB probe (PDF). Scheme S1, S2. Figure S1-S7. Table S1-S5.
AUTHOR INFORMATION Corresponding Author *Fax/Tel: +86-25-89687204, E-mail:
[email protected];
[email protected].
ACKNOWLEDGMENT This work is sponsored by the National Natural Science Foundation of China (No. 21501146 and 21335504), and the Natural Science Foundation of Jiangsu Province (No. BK20150424).
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II III I
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NaBH4
targeted imaging
Ag+ ASNCB cancer cell
multifunctional DNA
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